HK1234775B - Improved methods for processing dna substrates - Google Patents

Description

Improved methods for processing DNA substrates

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/934,515, filed on January 31, 2014, and U.S. Provisional Patent Application No. 62/078,309, filed on November 11, 2014, the disclosures of each of which are incorporated herein by reference in their entirety.

Electronic Submissions Incorporated by Reference

This application contains a sequence listing in computer-readable form (file name: 47999A_Seqlisting.txt; created on January 29, 2015; 47,035 bytes), which is an independent part of the disclosure and is incorporated herein by reference in its entirety.

introduction

All commercially available next-generation sequencing (NGS) technologies require library preparation, whereby specific adapter sequence pairs are ligated to the ends of DNA fragments to allow sequencing by the instrument. Most NGS adapters contain three functional domains: (1) a unique PCR primer annealing sequence for library and clonal amplification, (2) a unique sequencing primer annealing sequence, and (3) a unique sample index sequence. Currently, most platforms use clonal amplification to generate hundreds of copies of each individual DNA library molecule. This is achieved through bridge amplification or emulsion PCR, the purpose of which is to amplify the signal generated for a specific sequence detection mode (e.g., fluorescence or pH) for each library molecule. For sequencing by synthesis, the annealing domains of the sequencing primers are juxtaposed at the adapter insert junction; to allow paired-end sequencing, each adapter has a unique sequence for primer annealing. The sample index sequence contains a short unique sequence, typically 6-8 bases, that identifies the sample source of a specific sequence read when sequenced, allowing multiple sequencing or co-sequencing of samples. There are existing and emerging single molecule sequencing technologies that do not rely on clonal amplification for signal detection, but still require the attachment of adapter sequences to their ends for other purposes, for example, adding terminal hairpin loops to DNA duplexes to allow sequencing of both strands as a single molecule, or introducing leader sequences for nanopore entry.

Targeted next-generation sequencing is encompassed by two leading technologies: amplicon sequencing and target hybridization-capture enrichment of whole-genome libraries. Amplicon sequencing is the method of choice for rapid turnaround times, as it reduces the number of steps required, requires a significantly smaller set of target loci than a whole exome, and offers significant savings in overall costs, both in terms of preparative reagents and sequencing depth. Amplicon sequencing is represented by a variety of available technologies. Examples of these include 1. multiplex PCR, which uses degradable target-specific primers to eliminate primer dimers, followed by polishing and NGS adapter ligation, where overlapping targets are separated into separate tubes (Ion Torrent AmpliSeq); 2. multiplex extension-ligation reactions, which introduce NGS adapters to the ends of each target-specific oligonucleotide pair, followed by NGS adapter-mediated PCR amplification, which generally avoids multiplex PCR; however, ligation-mediated PCR requires higher amounts of input DNA (Illumina TSCA); 3. multiplex PCR performed on microfluidic chambers, which separates primer pairs to avoid primer dimer formation and allows overlapping target loci; the separate reactions require higher amounts of input DNA (Fluidigm Access Array); 4. multiplex PCR performed by digital droplet PCR, which also separates primer pairs to avoid primer dimer formation and allows overlapping target loci; again, it requires higher amounts of input DNA (Raindance). Each technology is designed to eliminate primer dimers or avoid their formation during multiplex amplification in order to avoid the resulting NGS amplicon library being dominated by these artifacts. The disadvantages of existing methods are: A. the high cost of microfluidic or digital droplet instruments and consumables, B. higher input requirements; and C. the need to separate multiple reactions, where overlapping or continuous coverage is required, thereby further increasing the input requirements. When continuous coverage is required, an alternative to these options is to perform long-fragment PCR. However, long-fragment PCR is difficult to multiplex, and the subsequent fragmentation required for most sequencing platforms after separation of NGS library preparation is both time-consuming and costly. The art needs a simple method for amplicon generation that allows low input of approximately 10 nanograms (ng) of DNA, does not require instruments other than thermal cyclers, and, when continuous coverage is required, is irrelevant whether the target is a separate hotspot locus or whether the target is an overlapping region of the genome. The compositions and methods described herein provide a solution that meets this need.

Typically, preparation of NGS DNA libraries involves five steps: (1) DNA fragmentation, (2) polishing, (3) adapter ligation, (4) size selection, and (5) library amplification (see Figures 1 and 2).

(1) Fragmentation: DNA fragmentation can be performed by enzymatic digestion or physical methods, such as sonication, nebulization or hydrodynamic shearing. Each fragmentation method has its advantages and limitations. Enzymatic digestion produces DNA ends, which can be efficiently polished and connected to the adapter sequence. However, it is difficult to control the enzymatic reaction and produce fragments with predictable lengths. In addition, enzymatic fragmentation is often base-specific, thus introducing representation bias into sequence analysis. Physical methods that make DNA fragmentation more random and the DNA size distribution may be easier to control, but the DNA ends produced by physical fragmentation are damaged, and conventional polishing reactions are insufficient to produce fully ligation-compatible ends.

(2) Polishing: A typical polishing mixture contains T4 DNA polymerase and T4 polynucleotide kinase (PNK). The 5'-3' polymerase and 3'-5' exonuclease activities of T4 DNA polymerase cut off 3' protrusions and fill in 3' recessed ends, which results in the excision of damaged 3' bases and polishing (generating blunt ends) the DNA ends. The T4 polynucleotide kinase in the polishing mixture adds phosphate to the 5' ends of DNA fragments, which may be deficient in this, thereby making them compatible with NGS adapter ligation.

What is still unknown in the art is that a large number of 5' ends produced by physical fragmentation are destroyed in an unknown manner and are not phosphorylated by PNK. There is no enzyme in the conventional finishing mixture that can trim the destroyed 5' terminal base. Thus, a large number of DNA fragments in the preparation are not converted into NGS library molecules because their 5' ends still remain incompatible with the NGS adapter connection. Although it is known in the art that adapter connection is invalid, the connection is usually carried out simultaneously on two chains, so it is still unknown which chain is limited. We divide the reaction into chain-specific connections to test their respective efficiency respectively. Through this analysis, we can find out the rate-limiting step in the overall process for the 5' end, which is a poor substrate for PNK and adapter connection for a large number of DNA fragments.

(3) Adapter ligation: In addition to the lack of 5' phosphate groups, another factor that contributes to low NGS library yield is the ligation reaction itself. Prior to ligation, adenylation of the repaired DNA using a DNA polymerase lacking 3'-5' exonuclease activity is typically performed to minimize chimera formation and adapter-adapter (dimer) ligation products. In these methods, a single 3' A-protruding DNA fragment is ligated to a single 5' T-protruding adapter, and the A-protruding fragment and the T-protruding adapter have sticky ends that are incompatible for self-ligation. However, the adenylation reaction is incomplete and produces non-specific byproducts, which further reduces the number of molecules available for ligation, thereby reducing library generation. More efficient alternative methods are provided herein to minimize concatemer formation.

(4) Size selection: The size selection process also affects library generation. During the size selection process, fragments of unwanted sizes are eliminated from the library by gel- or bead-based selection to optimize the library insert size for the desired sequencing read length. This maximizes sequence data output by minimizing the overlap of paired-end sequencing that occurs from short DNA library inserts. In cases where the sample has extremely limited input, this step can be skipped and, in exchange for a higher degree of paired-end overlap, more rare fragments can be sequenced.

(5) Amplification: The problem of low library yield necessitates PCR amplification of the library prior to NGS analysis, which results in a loss of library complexity and the introduction of base composition bias. Currently, the only solution to circumvent this problem is to use a higher amount of input DNA for library preparation, but up to 20% of clinical samples submitted for NGS analysis have insufficient DNA, so additional PCR cycles are performed instead to overcome the insufficient DNA input. This results in reduced sequence data due to an unacceptable percentage of PCR duplications.

Summary of the Invention

In order to solve the existing problem of low yield that causes NGS library construction described above, an enhanced adapter connection method is provided. This novel method overcomes the necessity of adding a phosphate group to the 5' end of DNA fragments (this is required for conventional adapter connection; see Figures 1 and 2). As an alternative, the 5' terminal base of the damage caused by the physical fragmentation of DNA is removed. By removing the base of the damage, the connection compatibility base with 5' phosphate is exposed, and the adapter connection efficiency is restored, which leads to a significant increase in library output and the ability to build a library from the amount of reduced input DNA. In addition, a substitute for the adenylylation/TA connection that prevents chimeric library inserts (multiplex formation in the connection process) and forms adapter dimer connection products is introduced, which also facilitates higher library preparation output. In any embodiment of the method described herein, the processing includes converting the 5' and/or 3' ends of the substrate molecule into a connection compatibility form.

The method, in its exemplary form, comprises four separate incubations (see Figures 3, 4, and 5) to produce processed substrate molecules. In the first incubation, double-stranded fragmented DNA is combined with a phosphatase under appropriate reaction conditions, which removes phosphate groups from the ends of the DNA fragments. This prevents the generation of chimeric library inserts by preventing the formation of DNA fragment concatemers in subsequent ligation reactions.

In the second incubation, the dephosphorylated DNA fragments are combined with a polymerase or polymerase mixture having 3'-5' exonuclease activity. Under appropriate reaction conditions and in the presence of dNTPs, the damaged 3' bases generated during the physical fragmentation process are corrected by excising the 3' protrusions and filling the 3' recessed ends, thereby achieving finishing of the double-stranded DNA fragments. Upon completion of this step, the DNA fragments have blunt ends with ligation-compatible 3' and 5' ends lacking phosphate groups, thereby preventing the DNA fragments from self-ligating.

In the third incubation, the blunt-ended double-stranded DNA fragments are combined with DNA ligase and a first double-stranded blunt-ended NGS adapter (3' adapter), wherein the first double-stranded blunt-ended NGS adapter comprises a 5' phosphate and is capable of connecting to the 3' end of the DNA fragment (see Figure 3). The special feature of the 3' adapter is that the adapter DNA chain (which would normally be connected to the 5' end of the DNA fragment at the same time) has a 3' end modification that prevents connection, so that after the connection reaction, even in the presence of 5' phosphate, a nick is retained at the junction of the 5' end of each DNA fragment and the 3' end of the 3' adapter. The same 3' modification that prevents connection to the 5' end of the DNA fragment can also prevent the formation of adapter-adapter connection products, although they will contain a single adapter sequence, which will not be a functional adapter dimer (functional dimer contains two adapters). The product of this step is a double-stranded DNA fragment with a single NGS adapter that is connected to only one chain of the two 3' ends.

In the fourth incubation, the chain of the 3' adapter that remains, which has not yet been connected to the DNA fragment (due to the 3' modification), is also displaceable or degradable, due to the introduction of degradable bases during oligomer synthesis. During the fourth incubation, in the presence of an optional, suitable enzyme, the 3' adapter chain is degraded or displaced (by a new single-stranded adapter), the new single-stranded adapter comprising a second NGS adapter sequence that is also present in the reaction (5' adapter, see Figure 3), and, through the complementary sequence of the 3' adapter at the junction of the adapter-insert, the single-stranded 5' adapter anneals to the complementary portion of the 3' adapter, which is connected to the 3' end of the double-stranded DNA fragment, which results in the recovery of the gap. In addition, in the case where the reaction is a DNA polymerase with 5'-3' exonuclease activity, and in the presence of dNTPs, a ligase and suitable reaction conditions, a nick translation is initiated at the gap or gap at the junction of the 5' adapter and the 5' end of the DNA fragment. The gap translation results in the replacement of the damaged 5' terminal base (and one or more other bases within the 5' end) and exposes the ligation-compatible 5' terminal phosphate group. Subsequently, when the ligase seals the gap and the gap is translated into one or more bases, efficient connection of the 5' adapter to the DNA substrate molecule occurs (see Figure 4). When the new adapter connection method is completed, the two ends of each double-stranded DNA fragment are flanked by two different single-stranded NGS adapters, and the different single-stranded NGS adapters share a short complementary adapter sequence located at the adapter insert junction.

Alternatively, removal of the 5' terminal base and ligation of the 5' adapter can be achieved without polymerization by annealing a single-stranded 5' adapter having one or more additional random bases at its 3' end that coincide with the damaged 5' base of the substrate molecule, and, in the absence of dNTPs, cleavage of the replaced base at the 5' end of the DNA substrate molecule by a 5' flanking specific nuclease following displacement, resulting in efficient ligation of the second NGS adapter to the exposed 5' phosphate on the end of the cleaved DNA substrate molecule (see Figure 5).

In another alternative, 5' terminal base removal and 5' adapter ligation can be achieved by extending from a single dideoxy base of a degradable or replaceable strand of the 3' adapter, followed by cleavage of the 5' terminal base of the DNA fragment by the 5' flank endonuclease activity of the polymerase. This strand is then degraded or replaced by the 5' adapter, and in the presence of a ligase, the 5' adapter is efficiently ligated to the exposed 5' phosphate on the DNA fragment. Alternative embodiments of this and the previous steps are shown below.

Thus, in one aspect, the present invention provides a method for producing a processed substrate molecule, the method comprising: (i) ligating a first polynucleotide to the 3' end of a substrate molecule, the substrate molecule being at least partially double-stranded; (ii) annealing a second polynucleotide to the first polynucleotide under conditions that promote annealing; (iii) cleaving at least one nucleotide from the 5' end of the substrate molecule; and then (iv) ligating the second polynucleotide to the 5' end of the double-stranded substrate molecule to produce a processed substrate molecule. In one embodiment, the method further comprises the step of, prior to step (i), contacting the substrate molecule with a phosphatase. In another embodiment, the method further comprises the step of causing the substrate molecule to have a blunt end by contacting the substrate molecule with a polymerase having 3'-5' exonuclease activity. In yet another embodiment, the method further comprises the step of contacting the substrate molecule with a template-independent polymerase to adenylate the 3' end of the substrate molecule.

In any of the methods described herein, it is envisioned that the substrate molecule is naturally occurring or that the substrate molecule is synthetic. In one embodiment, the substrate molecule is naturally occurring. In another embodiment, the substrate molecule is genomic DNA, and in another embodiment, the genomic DNA is eukaryotic or prokaryotic. In embodiments where the substrate molecule is genomic DNA, the present invention envisions that the genomic DNA is fragmented in vivo or in vitro. In some embodiments, the in vitro fragmentation is performed by a method selected from the group consisting of shearing, cleavage with an endonuclease, sonication, heating, irradiation with an α, β or γ source, chemical cleavage in the presence of metal ions, free radical cleavage, and combinations thereof. In some embodiments, the in vivo fragmentation occurs by a method selected from the group consisting of apoptosis, irradiation, and asbestos exposure.

The present invention also contemplates embodiments wherein the substrate molecule is synthetic and is selected from the group consisting of cDNA, DNA produced by whole genome amplification, primer extension products comprising at least one double-stranded end, and PCR amplicons.

In any aspect or embodiment of the invention, it is envisioned that the first polynucleotide is at least partially double-stranded and comprises oligonucleotide 1 and oligonucleotide 2. In some embodiments, the second polynucleotide anneals to oligonucleotide 1, and in another embodiment, the annealing results in a gap, a gap, or overlapping bases between the second polynucleotide and the substrate molecule. In some embodiments, the annealing results in dehybridization of oligonucleotide 1 and oligonucleotide 2.

In various embodiments, the second polynucleotide is contacted with a polymerase, resulting in degradation of oligonucleotide 2.

The present invention also contemplates embodiments in which oligonucleotide 2 comprises bases susceptible to degradation, and the present invention further contemplates embodiments in which oligonucleotide 2 comprises a blocking group at its 3' end that prevents ligation. In some embodiments, the second polynucleotide comprises modified bases.

In another embodiment, the method of the present invention further comprises: (i) linking the third polynucleotide to the 3' end of another substrate molecule that is at least partially double-stranded; (ii) annealing the fourth polynucleotide to the third polynucleotide under conditions that promote annealing; (iii) cutting at least one nucleotide from the 5' end of the other substrate molecule; and, then (iv) linking the fourth polynucleotide to the 5' end of the other double-stranded substrate molecule to produce a processed another substrate molecule. In some embodiments, the first polynucleotide and the third polynucleotide are the same. In some embodiments, the second polynucleotide and the fourth polynucleotide are the same.

The method for targeted amplicon NGS library construction includes two separate steps: multiplex PCR target enrichment followed by an NGS adapter ligation step (see Figure 39). Two separate workflow options are possible: two-step PCR followed by adapter ligation or one-step PCR followed by ligation.

In the multiplex PCR step using any of the methods, target-specific primer pairs are designed according to the desired target loci and contain universal truncated NGS adapter sequences at their 5' ends (see Figures 40 and 41, Table 2). The first PCR cycle has an extended cycle time to allow for high complexity of primer pairs, each of which is at a low concentration to generate amplicons with universal NGS adapter tags from their target sequences. These primers optionally have unique degenerate sequence tags to identify individual amplicons (UI = unique identifier), where each UI is located between the universal NGS adapter sequence at the 5' end of each primer and the target-specific portion at the 3' end (and is represented as an extension of NNNN bases, Figures 40, 41). If a UI sequence is used, the extended multiplex PCR cycle is limited to 2 times to avoid incorporating additional UI sequences into copies of previously generated amplicons; if a UI sequence is not used, the extended multiplex PCR cycle can be performed for more than 2 cycles. The more restricted the number of target-specific cycles performed, the fewer the accumulated primer-dimer products, so the minimum number of multiple cycles feasible for the amount of input sample should be performed. After the multiple cycles (2 times or more), the amplification of the second stage with a shorter extension time is continued with PCR, wherein a single universal primer is used, which corresponds to the universal truncated NGS adapter of each target amplicon flanking the target amplicon. Compared to the target-specific primers, the universal primer is used at a relatively high concentration, wherein the total number of cycles is determined by the required library yield. The concentration of the target-specific primer is not enough to amplify the target, so the universal primer that cannot interact with itself takes charge of the amplification reaction, wherein no additional primer-dimer is formed. In addition, the primer dimers accumulated during the limited multiple cycles will be shorter than the required amplicon in length and will be subject to a stable secondary structure, which results in a less efficient amplification performed by a single universal primer. If a UI sequence is used, a multiple primer exonuclease I digestion or purification step needs to be performed before adding the universal primer to prevent the subsequent copy of the amplicon previously generated by the additional UI sequence tag. If a UI sequence is not employed, a universal primer can be added at the beginning of a reaction employing multiplex primers and can function after universal adapter-tagged amplicons are generated.

Another feature of the universal primer is that it optionally includes a cleavable base to allow downstream adapters to connect. It is not intended to be limiting, and the cleavable base may include deoxyuracil, RNA or deoxyinosine. Alternatively, the universal primer does not include a cleavable base, and the sequence is subsequently excised using a 5' exonuclease to allow adapters to connect (see Figure 42). In addition, the two target-specific primers and the universal primer optionally include nuclease resistance modifications at their 3' ends; these include phosphorothioate connections, 2'O-methyl or methylphosphonate modifications. When a proofreading polymerase with 3' to 5' exonuclease activity is employed, these allow for more specific and efficient primer functions. If the enzyme is employed to remove the universal adapter sequence from the amplicon before the adapter is connected, it also limits 5' exonuclease digestion. After PCR, a purification step is required to remove unused reagents and polymerase.

For the final step of adapter ligation (see Figure 42), the portion of each amplicon derived from the universal primer is digested due to the inclusion of degradable bases in the primer and the use of modification-specific nucleases. Alternatively, for primers that contain nuclease-resistant bases at their 3' ends, the 5' portion of each amplicon can be trimmed by 5' exonuclease digestion. In this case, the exonuclease digestion of the 5' end of the amplicon will stop at the position of the nuclease-resistant base. The primer digestion reaction produces single-stranded 3' overhangs at both ends of each amplicon. Co-present in the reaction is a full-length, single-stranded adapter B, which contains a second NGS adapter sequence, and the single-stranded second adapter B anneals to the complementary portion of the universal adapter located at the 3' overhang of each amplicon through the complementary sequence of the universal adapter located at the adapter-target junction, wherein the adapter annealing results in the formation of a gap or gap. In addition, in the case where the reaction is a DNA polymerase with 5'-3' exonuclease activity, and in the presence of dNTPs, ligase and suitable reaction conditions, a gap translation is initiated at the gap or gap at the junction of the 5' end of the amplicon and the adapter B. The gap translation results in the replacement of one or more bases inside the 5' end and exposes the 5' terminal phosphate group of connection compatibility. Subsequently, when the ligase seals the gap, when the gap is translated into one or more bases, efficient connection of the adapter B to the DNA substrate amplicon occurs. Alternatively, the connection of the adapter B is completed by displacement-cleavage reaction, which adopts a polymerase with flanking nuclease activity, and additionally, ligase. In this case, dNTPs are not needed, and only several bases between the 3' end of the adapter B and the 5' end of the universal adapter part overlap and remain on each amplicon. In order to complete the adapter connection process, a joint-mediated connection is carried out simultaneously, so that the first adapter (A) on the remaining universal adapter sequence remaining at the 3' end of each amplicon is complete. The adapter oligonucleotide is complementary to the 3' end of the remaining universal adapter on each amplicon and is complementary to the oligonucleotide comprising the remainder of the first adapter. Through its complementarity for the two sequences, the adapter oligonucleotide hybridizes to the 3' remainder of the first adapter and the remaining universal adapter present on each amplicon, thereby allowing connection to occur. When the novel adapter connection method is completed, the two ends of each amplicon are flanked by two different, single-stranded NGS adapters (A and B), which have a short complementary adapter sequence located at the adapter target junction. Then, before library quantification and sequencing, a final purification step is performed.

Another feature of the methods disclosed herein is the selection of DNA polymerases used in the multiplex PCR amplification reactions. When high-fidelity Pfu DNA polymerase, Phusion DNA polymerase, KAPA HiFi DNA polymerase, Q5 DNA polymerase, or derivatives and analogs thereof are used, the error rate during the amplification process can be improved. Furthermore, given that the universal primers used in the second-stage amplification reaction optionally contain cleavable bases, high-fidelity DNA polymerases that tolerate uracil, RNA, or inosine bases are also desirable. This includes, but is not limited to, KAPA HiFi U+ polymerase, Themo Phusion U, and Enzymatics VeraSeq ULtra polymerase, all of which are engineered to tolerate uracil-containing substrates. Given that high-fidelity enzymes with 3' to 5' exonuclease activity are used in the amplification reactions, all target-specific primers and universal primers contain nuclease-resistant linkages at their 3' ends to improve the fidelity and efficiency of primer extension. This includes, but is not limited to, phosphorothioate linkages or other nuclease-resistant moieties.

In addition, as mentioned above, a method for multiplex PCR of a targeted NGS library capable of amplifying overlapping targets for continuous coverage in a single tube format is desired. The method disclosed herein can achieve this effect (Figures 43 and 44). In the case of two primer pairs with overlapping target regions, four possible amplicons can be produced: an amplicon specific for each of the two primer pairs, a large amplicon produced by the amplification of the two distal primers, and a small amplicon produced by the amplification of the two proximal primers. In order to avoid allowing small amplicons to dominate the multiplex PCR reaction (for example, the short amplicon and primer dimers often dominate the amplification reaction due to their shorter length and easy amplification properties), most methods divide the overlapping primer pairs into two tubes, which is effective but doubles the workload and the required DNA input amount. The method described herein allows overlapping amplicons to be produced in a single tube because, in view of the presence of universal sequences at each end, the short amplicon will be subject to a stable secondary structure, which results in a less efficient amplification by a single universal primer. Therefore, even if a small amplicon is produced during the initial target-specific PCR cycle, it will not be amplified efficiently. Thus, using the methods described herein, only amplicons specific for each primer pair and mega-amplicons are generated from high-quality, high-molecular-weight DNA input. When cross-linked FFPE DNA or fragmented DNA (particularly circulating cell-free DNA in the 165 bp range) is used, the formation of mega-amplicons is suppressed because the template length or integrity cannot support an amplicon of that size, and only amplicons specific for each primer pair are generated.

In any of the methods described herein, it is contemplated that the sample DNA input is naturally occurring. In one embodiment, the input DNA is genomic DNA, either intact high molecular weight DNA or fragmented circulating cell-free DNA, while in another embodiment, the genomic DNA is of eukaryotic, prokaryotic, mitochondrial, or viral origin. In other embodiments, the input DNA is single-stranded or double-stranded, or is synthetic and is derived from prior whole genome amplification or from random or other guided RNA reverse transcription.

In another aspect of the invention, a composition is provided comprising a ligase and a first polynucleotide that is at least partially double-stranded and comprises oligonucleotide 1 and oligonucleotide 2; wherein oligonucleotide 1 comprises a 5' phosphate and a blocking group at its 3' terminus; and wherein oligonucleotide 2 (i) comprises a base that is susceptible to degradation, and/or (ii) can be displaced by non-denaturing heat conditions, and further comprises a blocking group at its 3' terminus that prevents ligation of the 3' terminus of oligonucleotide 1 but allows ligation of the 5' terminus.

In some embodiments, the 3' blocking group of oligonucleotide 2 is 3' deoxythymine, 3' deoxyadenine, 3' deoxyguanine, 3' deoxycytosine, or a dideoxynucleotide. In another embodiment, the base susceptible to degradation is deoxyuracil, a ribonucleotide, deoxyinosine, or inosine. In various embodiments, the non-denaturing heat conditions are from about 50°C to about 85°C.

In some embodiments, oligonucleotide 2 comprises a base modification that reduces the binding stability of oligonucleotide 2, wherein the base modification is deoxyinosine, inosine, or a universal base.

In some aspects, the present invention also provides a composition comprising: a ligation product produced by incubating a double-stranded substrate with the composition of the present invention; a ligase, a DNA polymerase having nick translation activity, a nuclease that recognizes bases that are susceptible to degradation, and a second polynucleotide, which is single-stranded and comprises a 3' domain that is sufficiently complementary to the 5' portion of oligonucleotide 1 of polynucleotide 2 to anneal under appropriate conditions when oligonucleotide 2 of polynucleotide 1 is degraded or displaced.

In some embodiments, the second polynucleotide has sufficient length to replace the position of oligonucleotide 2 of the first polynucleotide, or the second polynucleotide comprises a base modification that increases its binding stability. In other embodiments, the endonuclease is selected from the group consisting of UDG plus endonuclease VIII, RNase HI, RNase H2, and endonuclease V. In other embodiments, the ligase is E. coli DNA ligase or T4 DNA ligase. In various embodiments, the base modification that increases its binding stability is locked nucleic acid (LNA).

In another aspect of the present invention, a composition is provided, comprising a ligation product produced by incubating a double-stranded substrate with a composition of the present invention; a ligase; a flanking endonuclease; an endonuclease that recognizes bases that are susceptible to degradation; and a second polynucleotide, wherein the second polynucleotide comprises a single-stranded oligonucleotide containing a 3' domain that is sufficiently complementary to the 5' portion of oligonucleotide 1 of polynucleotide 2 to anneal under appropriate conditions when oligonucleotide 2 of polynucleotide 1 is degraded or replaced, wherein the second polynucleotide has a length sufficient to replace the position of oligonucleotide 2 of the first polynucleotide, or the second polynucleotide comprises a base modification that increases its binding stability, and wherein the second polynucleotide further comprises a 3' terminal degenerate base.

In another aspect, the present invention provides a method for producing a processed substrate molecule, the method comprising: (i) ligating a first polynucleotide to the 3' end of a substrate molecule, wherein the substrate molecule is at least partially double-stranded; (ii) annealing a second polynucleotide to the first polynucleotide under conditions that promote annealing; (iii) cleaving at least one nucleotide from the 5' end of the substrate molecule; and then (iv) ligating the second polynucleotide to the 5' end of the double-stranded substrate molecule to produce a processed substrate molecule. In some embodiments, the method further comprises the step of contacting the substrate molecule with a phosphatase prior to step (i).

In some embodiments, the phosphatase is calf intestinal phosphatase or shrimp phosphatase.

In other embodiments, the method further comprises the step of making the substrate molecule have a blunt end by contacting the substrate molecule with a polymerase having 3'-5' exonuclease activity.

In some embodiments, the polymerase is selected from the group consisting of T4 DNA ligase, Klenow fragment, T7 polymerase, and combinations thereof. In another embodiment, the method further comprises the step of contacting the substrate molecule with a template-independent polymerase to adenylate the 3' end of the substrate molecule.

In various embodiments, the substrate molecule is naturally occurring or the substrate molecule is synthetic. Thus, in some embodiments, the substrate molecule is naturally occurring. In other embodiments, the substrate molecule is genomic DNA. In yet other embodiments, the genomic DNA is eukaryotic or prokaryotic, and in yet other embodiments, the genomic DNA is fragmented in vivo or in vitro. In some embodiments, the substrate molecule is circulating cell-free DNA.

In some embodiments, the method further comprises, before step (i), adjusting the temperature to about 50° C. to about 85° C. In some embodiments, the temperature is 65° C.

In other embodiments, the in vitro fragmentation is performed by a method selected from the group consisting of shearing, cleavage with an endonuclease, sonication, heating, irradiation with an alpha, beta, or gamma source, chemical cleavage in the presence of metal ions, free radical cleavage, and combinations thereof. In other embodiments, the in vivo fragmentation occurs by a method selected from the group consisting of apoptosis, irradiation, and asbestos exposure.

In other embodiments, the substrate molecule is synthetic and is selected from the group consisting of cDNA, DNA produced by whole genome amplification, primer extension products comprising at least one double-stranded end, and PCR amplicons.

In some embodiments, the first polynucleotide is at least partially double-stranded and comprises oligonucleotide 1 and oligonucleotide 2. In various embodiments, the second polynucleotide is annealed to oligonucleotide 1. In some embodiments, the annealing results in a gap, a gap, or overlapping bases between the second polynucleotide and the substrate molecule.

In various embodiments, the second polynucleotide is contacted with a polymerase, resulting in degradation of oligonucleotide 2.

In some embodiments, oligonucleotide 2 comprises a base that is prone to degradation. In other embodiments, the base that is prone to degradation is selected from the group consisting of deoxyuracil, RNA, deoxyinosine, and inosine. In other embodiments, oligonucleotide 2 comprises a blocking group at its 3' end that prevents connection. In different embodiments, the blocking group is a 3' deoxynucleotide or a dideoxynucleotide.

In some embodiments, the second polynucleotide comprises modified bases.

In other embodiments, annealing results in dehybridization of Oligonucleotide 1 and Oligonucleotide 2.

In yet another embodiment, the method further comprises: (i) ligating a third polynucleotide to the 3' end of another substrate molecule that is at least partially double-stranded; (ii) annealing a fourth polynucleotide to the third polynucleotide under conditions that promote annealing; (iii) cutting at least one nucleotide from the 5' end of the another substrate molecule; and, then (iv) ligating the fourth polynucleotide to the 5' end of the double-stranded another substrate molecule to produce a processed another substrate molecule.

In some embodiments, the first polynucleotide and the third polynucleotide are identical. In other embodiments, the second polynucleotide and the fourth polynucleotide are identical.

In another aspect, the present invention provides a composition comprising a universal primer and a plurality of target-specific oligonucleotide primer pairs; wherein each target-specific primer of the plurality of primer pairs comprises a target-specific sequence and a 5' terminal sequence that is not complementary to a target substrate molecule; wherein the universal primer comprises the 5' terminal sequence and a cleavable base or a nuclease-resistant modification; wherein each target-specific primer of the plurality of primer pairs and the universal primer each comprises a nuclease-resistant modification at its 3' terminus; a high-fidelity polymerase that tolerates the introduction of the cleavable base into the universal primer; wherein the target-specific primer pairs and the universal primer anneal to their target substrate molecules at the same temperature; and wherein the molar ratio of target-specific primer to universal primer is at least about 1:100.

In some embodiments, the cleavable base is deoxyuracil, RNA, deoxyinosine, or inosine. In other embodiments, the nuclease-resistant modification is a phosphorothioate.

In other embodiments, at least one target-specific primer further comprises a molecular identification tag between the target-specific sequence and the 5' end sequence.

In various embodiments of the invention, the molar ratio of target-specific primers to universal primers is at least about 1:200, or at least about 1:300, or at least about 1:400, or at least about 1:500, or at least about 1:1000, or at least about 1:2000, or at least about 1:3000, or at least about 1:5000, or at least about 1:10,000 or more.

In various embodiments, the composition further comprises a substrate molecule.

In some aspects, a composition is provided comprising a polymerase chain reaction (PCR) product produced by a universal primer, wherein the product comprises at least one cleavable base introduced by the universal primer; a nuclease capable of cleaving the cleavable base; (i) at least one nucleotide and a DNA polymerase with nick translation activity, or (ii) an enzyme with flanking nuclease activity; a DNA ligase; a 5' adapter, wherein the 5' adapter comprises (i) a 3' sequence that is complementary to the 5' portion of the reverse complement sequence of the universal primer exposed by the nuclease cleavage of the universal primer, and (ii) a 5' portion that is not complementary to the reverse complement sequence of the universal primer; and wherein the 3' portion of the reverse complement sequence of the universal primer anneals to a partially double-stranded truncated 3' adapter.

In some embodiments, the endonuclease is selected from the group consisting of UDG+Endonuclease VIII, RNase HI, RNase H2, and Endonuclease V. In other embodiments, the DNA ligase is E. coli DNA ligase or T4 DNA ligase.

In another aspect, the present invention provides a composition comprising: a polymerase chain reaction (PCR) product produced by a universal primer, wherein the product comprises at least one nuclease-resistant modification introduced by the universal primer; a 5' exonuclease, wherein the 5' exonuclease is unable to digest the PCR product beyond the nuclease-resistant modification; (i) at least one nucleotide and a DNA polymerase with nick translation activity, or (ii) an enzyme with flanking endonuclease activity; a DNA ligase; a 5' adapter, wherein the 5' adapter comprises (i) a 3' sequence that is complementary to the 5' portion of the reverse complement sequence of the universal primer exposed by endonuclease cleavage of the universal primer, and (ii) a 5' portion that is not complementary to the reverse complement sequence of the universal primer; and wherein the 3' portion of the reverse complement sequence of the universal primer anneals to a partially double-stranded truncated 3' adapter molecule.

In yet other aspects, a method for polymerase chain reaction (PCR) is provided, the method comprising contacting a substrate molecule with: (i) a target-specific primer pair, wherein each primer comprises a 5' sequence that is not complementary to the substrate molecule and introduces a single universal adapter at the end of the resulting amplicon; and (ii) a single primer, wherein the single primer comprises the single universal adapter sequence and further comprises a cleavable base or a nuclease-resistant modification, wherein, under suitable reaction conditions, in the presence of a high-fidelity DNA polymerase and nucleotides, each cycle of PCR employs a constant annealing temperature but a different annealing time, wherein the molar ratio of each target-specific primer to the universal primer is at least about 1:100, and target-specific amplicons are produced during the first two or more PCR cycles having an annealing time of 5 minutes or longer, and the resulting amplicons are subsequently amplified in the remaining PCR cycles, wherein the remaining PCR cycles each comprise an annealing time of 1 minute or less, wherein amplification of the target-specific amplicons is achieved by a higher concentration of the single universal primer.

In other aspects, a method for multiplex PCR is provided, comprising: contacting a substrate molecule with: (i) multiple target-specific primer pairs, wherein each primer comprises a 5' sequence that is not complementary to the substrate and introduces a single universal adapter at the end of the resulting amplicon, and (ii) a single primer, wherein the single primer comprises the single universal adapter sequence and further comprises a cleavable base or a nuclease-resistant modification, wherein, under appropriate reaction conditions, in the presence of a high-fidelity DNA polymerase and nucleotides, each PCR cycle adopts a constant annealing temperature but applies a different annealing time, wherein the molar ratio of each target-specific primer: universal primer is at least about 1:100, and the target-specific amplicons are produced during the first two or more PCR cycles having an annealing time of 5 minutes or longer, and the resulting amplicons are subsequently amplified during the remaining PCR cycles, wherein the remaining PCR cycles each comprise an annealing time of 1 minute or less, wherein multiple amplification of the target-specific amplicons is achieved by a higher concentration of the single universal primer.

In some aspects, a method is provided for converting polymerase chain reaction (PCR) products comprising converting a single universal adapter sequence at each end into a product comprising an asymmetric 5' and 3' adapter at each end, the method comprising: (a) digesting the 5' end of the PCR product, wherein a cleavable base or nuclease-resistant modification is introduced, followed by (b) annealing and (i) gap translation ligation or (ii) flanking endonuclease cleavage ligation of a 5' adapter that is complementary to the 5' portion of the reverse complement sequence of the universal adapter exposed by digestion, and (c) wherein a partially double-stranded truncated 3' adapter is annealed and ligated to the 3' portion of the reverse complement sequence of the universal adapter, thereby converting the PCR product into a product comprising an asymmetric adapter at each end thereof.

In some embodiments, the PCR product is a whole genome amplification (WGA) product.

In any of the methods described herein, it is contemplated that the target loci selected for multiplex amplification correspond to any of a wide variety of applications, including but not limited to oncology-specific targets, drug resistance-specific targets, targets for genetic diseases, targets from infectious pathogens, targets for pathogen hosts, species-specific targets, and any clinically useful target.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Existing NGS adapters

Filler adapters (blunt ends or with T-overhangs) have 3' and 5' hydroxyl groups

Y-adapter (with T-overhang) has 3' hydroxyl group and 5' phosphate group

Stem-loop adapters (blunt ends or with T-overhangs) have 3' hydroxyl and 5' hydroxyl or phosphate

Figure 2, A and B. Conventional adapter ligation chemistry

Figure 3 Characteristics of 3' and 5' adapters

Figure 4 5' adapter ligation via nick translation

The steps include:

-Dephosphorylation of substrate molecules

-Substrate molecule finishing/blunt end generation

-3' adapter ligation

- Partial degradation of the 3' adapter and annealing of the 5' adapter

-Polymerase extension of the 5' adapter by nick translation

- Ligation of the extended 5' adapter to the exposed 5' phosphate of the DNA substrate

Figure 5 5' adapter ligation via displacement-cleavage

The steps include:

1 – Dephosphorylation of substrate molecules

2-Substrate molecule end finishing/blunt end generation

3–3’ adapter ligation

Partial degradation of the 4–3’ adapter and annealing of the 5’ adapter

5 – Replacement of one or more 5’ bases of a DNA fragment and annealing of one or more 3’ bases of a 5’ adapter

6 – Cleavage of DNA by 5'-flanking endonucleases that replace one or more 5' bases

Ligation of the 3’ end of the 7–5’ adapter to the exposed 5’ phosphate of the substrate DNA

Figure 6: Adapter ligation is completed using two incubations

5'-adapter attachment is achieved by coupled annealing-nick translation-ligation in two incubations, where the first incubation is 3'-adapter ligation and the second incubation combines three reactions that occur sequentially: (1) annealing of the 5'-adapter, (2) extension of the 5'-adapter by a DNA polymerase with nick translation activity (removing the damaged 5' end of the substrate DNA), and (3) ligation of the 5'-adapter to the exposed 5'-phosphate of the substrate DNA.

5'-adapter ligation by coupled annealing-base excision-ligation is achieved in two incubations, where the first incubation is 3'-adapter ligation and the second incubation combines three reactions that occur in sequence: (1) annealing of the 5'-adapter to one or several random bases at the 3' end and displacement of one or several terminal 5'-bases of the substrate DNA, (2) cleavage of the displaced 5' base by a 5'-flanking nuclease (which excises the damaged 5' end of the substrate DNA), and (3) ligation of the 5'-adapter to the exposed 5'-phosphate of the substrate DNA.

Figure 7 Generation of single-stranded 3' overhangs

The 3'-adapter overhang sequence can be enzymatically added by at least 4 different methods:

o By conventional ligation using T4 DNA ligase

o Through single-stranded DNA (RNA) ligase

o Conventional homopolymer tailed by terminal transferase

o Controlled tailing and simultaneous adapter ligation by employing terminal transferase, DNA ligase, and attenuator-adapter molecules. See International Patent Application No. PCT/US13/31104, filed March 13, 2013, which is incorporated herein by reference in its entirety.

Alternatively, DNA fragmentation or other processing may result in pre-existing DNA ends with 3'-overhangs sufficient for annealing of 5' adapters.

Figure 8. Method for annealing 5' adapters. Figure 8A depicts steps (i)-(iii); Figure 8B depicts steps (iv)-(v).

i) by ligation after degradation of the second oligonucleotide, which was previously annealed to the 3'-adapter

ii) by competitive displacement of a second oligonucleotide previously annealed to the 3'-adapter

iii) by binding to the upstream region of the 3'-adapter (followed by restricted nick translation and degradation of the second oligonucleotide previously annealed to the 3'-adapter)

iv) by pre-annealing the 5'-adapter to the upstream region of the 3'-adapter (followed by restricted nick translation and degradation of the second oligonucleotide, which was previously annealed to the 3'-adapter)

v) by replacing the second oligonucleotide with a 3'-blocked 5'-adapter, which is activated by cleavage

Figure 9A-D . 5' adapter ligation using single base extension

Figure 10 Synthesis of Illumina NGS Library 1

Library 1 synthesis occurs in 5 or 6 steps:

Figure 10a:

1 – Dephosphorylation and finishing of substrate molecules

Ligation of 2–3’ adapters to Illumina sequences P7 (a) or P5’ (b)

Partial degradation of the 3’ adapter and annealing of the complementary 5’ adapter to the Illumina sequence P5 (a) or P7’ (b).

4 – polymerase extension of the 5’ adapter via nick translation, and

Ligation of the 3’ end of the 5–5’ adapter to the exposed 5’ phosphate of the DNA substrate

Or, in Figure 10b :

4 – Replacement of one or more 5’ bases of the DNA substrate and annealing of one or more 3’ bases of the 5’ adapter

5-cleavage of the DNA substrate at the displaced 5' base(s) by a 5'-flanking endonuclease, and

Ligation of the 3’ end of the 6–5’ adapter to the exposed 5’ phosphate of the DNA substrate

The library was amplified by PCR using primers P5 and P7'

Figure 11 Synthesis of Illumina NGS Library II

Library II synthesis occurs in 4 steps:

1 - Synthesis of NGS libraries using truncated adapter P7 by one of the two methods described in Figure 6

2 – Library amplification using truncated or full-length degradable primer P7*

3 – Degradation of the incorporated P7* primer followed by annealing and ligation of the 5’ adapter P5

4- If the truncated degradable primer P7* is used in step 2, bridge-ligation of the P7*" adapter with the truncated adapter P7*' is performed to complete the full-length adapter P7

Figure 12 A and B. Synthesis of Ion Torrent Library

Library synthesis was performed as follows:

1 – DNA substrate dephosphorylation and finishing

Ligation of the 2–3’ adapter to the sequence A1’-P1’ (a) or A’ (b)

3 - Nick translation ligation or base cutting ligation of the 3' end of the 5' adapter with sequence A (a) or sequence P1-A ₁ (b) to the 5' end of the trimmed DNA

4 – Library amplification by PCR using primers A and P1

Figure 13 Synthesis of an Ion Torrent library with 96 combinatorial barcode sequences using only 20 adapter sequences

Library synthesis steps:

1 – DNA end dephosphorylation and polishing (not shown)

2 – Ligation of a (blunt) 3'-adapter P1 _n with the sequence T' _n -L'-P1' and a 5' phosphate group and a 3'-blocked complementary oligonucleotide with the sequence P1 _tr -LT _n

Degradation of 3-3'-blocked complementary oligonucleotide P1 _tr -LT _n

4—5'-Adapter _Am with sequence _Atm -L anneals to linker region L'

5 – Extension of the 5'-adapter _Am by nick translation polymerization and ligation of the 3' end of the extended 5'-adapter _Am to the 5' end of the DNA

6 – Library amplification by PCR using primers A and P1

The adapters with combinatorial barcodes include: 8 adapters P1 _n , which contain barcode sequences T ₁ , T ₂ , ..., T ₈ , and 12 adapters A _m , which contain barcode sequences t ₁ , t ₂ , ..., t ₁₂

The resulting library has combinatorial barcode sequences _tm - _LTn with up to 96 barcode combinations.

Figure 14 Enrichment of selected restriction fragments by 5' adapter ligation

Restriction DNA fragments are selected by 5'-adapter ligation followed by PCR amplification. Selection occurs via two 5'-adapters - selectors A and B, which contain sequences a and b identical to the 5' end sequences of the restriction fragments. The enrichment method involves:

1. DNA digestion using restriction endonucleases;

2. Ligation of 3'-adapter;

3. Partial degradation of the 3'-adapter and annealing of the 5'-adapter-selector;

4.5'-Adapter-Selector invades the terminal sequences a and b of the restriction fragment;

5. The displaced terminal sequences a and b are cleaved by the 5'-flanking endonuclease;

6.5'-Adapter-Selector Ligation to the Ends of the Restriction Fragments;

7. Amplify the selected restriction fragments by PCR.

Steps 1, 2, and steps 3–6 can be combined into a single incubation reaction.

Figure 15 Target enrichment by primer extension

Enrichment is performed by 5' adapter ligation, where the 3' overhang is generated by primer extension using adapters A and B, which are complementary to the target DNA region on the library, and partial digestion of the 5' domain of adapter A. The biotinylated 5'-adapter is annealed to the 3'-overhang of adapter A and then ligated to the 5' end of adapter A after trimming by limited nick translation (a) or invasion-cleavage reaction (b). Library fragments containing the target DNA region are then isolated by affinity capture using streptavidin magnetic beads, amplified by PCR, and analyzed by sequencing.

Figure 16 Alternative library construction 1

Library construction can be performed in 6 or 7 steps using a single adapter:

1 – Dephosphorylation of substrate molecules

2-Substrate molecule end finishing/blunt end generation

Ligation of the 3–3’ adapter to sequence A’

Partial degradation of the 4–3’ adapter and annealing of the complementary 5’ adapter to sequence A

5 – polymerase extension of the 5’ adapter via nick translation, and

Ligation of the 3’ end of the 6–5’ adapter to the exposed 5’ phosphate of the DNA substrate

Alternatively, through

5 – Replacement of one or more 5’ bases of DNA and annealing of one or more 3’ bases of 5’ adapter

6 – cleavage of the DNA by a 5'-flanking endonuclease at the replaced 5' base or bases, and

Ligation of the 3’ end of the 7–5’ adapter to the exposed 5’ phosphate of the DNA substrate

The library can be amplified by PCR using a single primer A

Figure 17 Alternative library construction II

Adapter ligation can generate a library of double-stranded DNA fragments with covalently linked 3' and 5' DNA ends. Library construction is performed as follows:

1 – Dephosphorylation of substrate molecule (not shown)

2-Substrate molecule end finishing/blunt end generation (not shown)

3—Ligation of a hairpin blunt-end adapter with a phosphorylated 5' end and a blocked (optional) 3' end

4 – Partial degradation of the hairpin adapter to generate an extendable 3’ end

Nick translation of the 3' end of the 5-hairpin adapter and its ligation to the exposed 5' phosphate of the DNA substrate

Figure 18 Alternative library construction III

A circularized NGS library can be constructed using the following steps:

1 – Dephosphorylation of substrate molecule (not shown)

2-Substrate molecule end finishing/blunt end generation (not shown)

3 - Ligation of adapters with phosphorylated 5' ends and blocked (optionally) 3' ends and complementary sequences X and X'

4 – Degradation of unligated adapter strands to generate single-stranded 3’ overhangs

5 – Non-covalent circularization of DNA by annealing of terminal sequences X and X' (performed at low DNA concentrations)

6-Covalent Circularization of DNA by Nick Translation Ligation

FIG19 Comparison of conventional adapter ligation and 3′ adapter ligation using a FAM-labeled oligonucleotide substrate (Example 1)

FIG20 Comparison of conventional adapter ligation and 3′ adapter ligation using sheared, size-selected genomic DNA substrate (Example 2)

Figure 21 A and B. Temperature optimization of 5' adapter ligation using FAM-labeled oligonucleotide substrate (Example 3)

Figure 22 Analysis of the effect of dNTP composition on 5' adapter ligation (Example 4)

Figure 23 A and B. Paired nick translation-ligation reactions using thermostable enzymes (Example 5)

Figure 24 Paired displacement-cleavage-ligation reaction (Example 6)

FIG25 Paired displacement-cleavage-ligation reactions using "N" universal/degenerate or "T" substrate-specific 5' adapter 3' overhangs (Example 7)

Figure 26 Paired nick translation-ligation reaction using DNA polymerase I (Example 8)

FIG27 Blunt-end ligation of physically sheared DNA requires polishing, and dephosphorylation prevents the formation of chimeric ligation products (Example 9)

FIG28 A and B. NGS libraries have increased yield when prepared using 5′ base trimming combined with adapter ligation (Example 10)

Figure 29A, B and C. Sequence analysis of NGS libraries prepared using 5' base trimming combined with adapter ligation (Example 11)

FIG30 depicts the structures of the adapter, model substrate, and oligonucleotide constructs described in Example 1.

Figure 31 depicts FAM substrate molecules (see Example 1).

Figure 32 - Describes the structure of the adapter described in Example 2

Figure 33 - Describes the oligonucleotide construct system described in Examples 3, 4, 5 and 8.

Figure 34 - Describes the oligonucleotide construction system described in Example 6.

Figure 35 - Describes the oligonucleotide construction system described in Example 7.

FIG. 36 —Description of the oligonucleotide construction system described in Example 7.

FIG. 37 —Depicts the structures of the P7 and P5 adaptors described in Example 10.

FIG. 38 —Depicts the structures of the P7 and P5 adaptors described in Example 11.

Figure 39. Depicting two workflows for amplicon NGS library construction methods.

Figure 40. Depicts the first workflow where multiplex PCR is separated by a purification step.

Figure 41. Describes the second workflow where multiplex PCR is performed as a single step.

Figure 42. Depicts the final step of simultaneous ligation of adapters A and B to each amplicon.

Figure 43. Comparison of single-tube and dual-tube workflows.

Figures 44A and 44B depict amplicon products generated by overlapping primer pairs targeting a region of interest.

Figure 45. Example 1: Amplicon coverage on TP53 coding exons.

Figure 46. Example 1: Identification of somatic mutations in exon 8 of TP53.

Detailed Description of the Invention

In one aspect, the present invention describes an efficient method for connecting an adapter to the end of a fragmented double-stranded DNA molecule. The DNA molecule is referred to herein as a "substrate molecule". In one aspect, the method comprises a single incubation comprising: (1) annealing a 5' adapter to a pre-existing 3' overhang on a substrate molecule, preferably a 3' adapter, (2) removing the damaged base from the 5'-end of the substrate molecule, which allows (3) efficient connection of the 5' adapter to the exposed 5'-phosphate of the substrate molecule. In another aspect, the method comprises two incubations, wherein in the first incubation, the 3' adapter is connected to the substrate molecule, and in the second incubation, the 5' adapter is connected to the substrate molecule, as described above (see Figure 6). In various embodiments, the present invention also provides methods that include additional steps occurring prior to one or both ligation steps, including: (i) a dephosphorylation reaction, (ii) a polishing reaction to remove the damaged 3' end and generate a blunt end, and (iii) an adenylation reaction; various combinations of these steps are contemplated by the present invention and are discussed in further detail below.

In another aspect, the present invention describes an efficient method for preparing a multiplex amplicon NGS library. In one aspect, the method allows for the synthesis and amplification of multiple overlapping amplicons in a single tube. In another aspect, it describes a novel, efficient method for connecting an adapter to the end of a PCR amplicon that does not contain chimeric amplicons and adapter-dimers. In one aspect, it allows the introduction of unique degenerate sequence tags to identify individual amplicons. In another aspect, the method comprises a single incubation, which includes degrading the 5' end of the amplicon, followed by simultaneous connection of the second adapter B and the connector-mediated connection of the remainder of the first adapter A to the substrate amplicon. In different embodiments, the present invention also provides a method comprising an additional step occurring before the connection step, comprising: (i) a multiplex PCR reaction (ii) a purification step, and (iii) a universal single primer amplification step. Alternatively, the additional step occurring before the connection step comprises: (i) a combined multiplex PCR reaction amplified using a universal single primer, followed by (ii) a purification step. The present invention contemplates different options for the steps and is discussed in further detail below.

As used herein, the term "reaction conditions" or "standard reaction conditions" refers to conditions as specified by the manufacturer. It should be understood that, unless otherwise indicated, all enzymes described herein are used under standard reaction conditions. As used herein, the term "first polynucleotide" can be used interchangeably with "3'adapter,""firstadapter," or "Adapter A," and as used herein, the term "second polynucleotide" can be used interchangeably with "5'adapter,""secondadapter," or "Adapter B." In some cases, when Adapter A is used in reference to the IonTorrent ^™ technology, e.g., Figures 12-13, this refers to the Adapter A provided by the manufacturer for use in the IonTorrent ^™ method, and not the "Adapter A" defined herein.

As used herein, a "3' adapter" is attached to the 3' end of a substrate molecule, while a "5' adapter" is attached to the 5' end of a substrate molecule.

As used herein, a "disrupted" 5' end is a 5' end that lacks a 5' phosphate.

As used herein, a "processed" substrate molecule is a substrate molecule to which a 5' adaptor has been attached.

As used herein, a "high-fidelity polymerase" is a polymerase that has 3'-5' exonuclease (ie, proofreading) activity.

As used herein, the term "tolerant" refers to the property of a polymerase that is able to extend through templates containing cleavable bases (eg, uracil, inosine, and RNA).

The term "asymmetric" as used herein refers to a double-stranded molecule having two adapters at both ends rather than a single adapter at both ends. Thus, the asymmetry arises from the two adapters being highly non-complementary to each other and having single-stranded portions.

As used herein, a "universal primer" is an oligonucleotide used in an amplification reaction to introduce a universal adapter sequence. As used herein, a "universal adapter" corresponds to the portion of the amplification product of the universal primer sequence and its reverse complement.

It is understood that modifications that reduce the stability of the binding of two nucleic acids include, but are not limited to, nucleotide mismatches, deoxyinosine, inosine, or universal bases.

It is also understood that modifications that increase the stability of the binding of two nucleic acids include, but are not limited to, locked nucleic acids (LNA), spermine and spermidine or other polyamines, and thymidine methylation.

As used herein, the term "universal base" is a base that can base pair with all four naturally occurring bases without hydrogen bonding and with low destabilization to mismatches, and includes, but is not limited to, 5' nitroindole.

As used herein, a "molecular identification tag" is 4 to 16 bases in length, with an optimal length of 8 to 12 degenerate N bases.

Substrate molecules

It is contemplated that the substrate molecule is obtained from a naturally occurring source, or may be synthetic. Naturally occurring sources include, but are not limited to, genomic DNA, cDNA, DNA produced by whole genome amplification, primer extension products comprising at least one double-stranded end, and PCR amplicons. In various embodiments, the naturally occurring source is a prokaryotic source or a eukaryotic source. For example, but not intended to be limiting, the source may be human, mouse, viral, plant, or bacterial, or a mixture comprising multiple genomes.

It will be understood that "amplicon" as used herein refers to a portion of a polynucleotide that has been synthesized using amplification techniques.

If the source of the substrate molecule is genomic DNA, it is envisioned in some embodiments that the genomic DNA is fragmented. Fragmentation of genomic DNA is a conventional process known to those skilled in the art and is performed in the following ways: for example, but not limited to, in vitro by shearing (nebulizing) DNA, cutting DNA with endonucleases, sonicating DNA, by heating DNA, by irradiating DNA with α, β, γ or other radioactive sources, by light, by chemically cutting DNA in the presence of metal ions, by free radical cutting, and combinations thereof. Fragmentation of genomic DNA can occur in vivo, for example, but not limited to, due to apoptosis, irradiation and/or asbestos exposure. According to the methods provided herein, the population of substrate molecules does not need to have a uniform size. Therefore, the methods of the present invention are effective in using populations of substrate polynucleotide fragments of different sizes.

In some embodiments, the substrate molecule is at least partially double-stranded and comprises a 3 ' protrusion (see Fig. 7 a), a blunt end, a 3 ' recessed end, or no 3 ' hydroxyl group. The length of the protrusion or recessed end of the substrate polynucleotide can vary. In different aspects, the length of the protrusion or recessed end of the substrate molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Nucleotide. In other embodiments, the length of the protrusion or recessed end of the substrate molecule is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 Nucleotide. In yet other embodiments, the length of the protruding or recessed end of the substrate molecule is about 1 to about 5, or about 1 to about 10, or about 1 to about 15, or about 1 to about 20 nucleotides. In various aspects, the population of substrate molecules includes those in which more than one of the aforementioned types of substrate molecules is present in a single reaction. The present invention also contemplates substrate molecules that are at least partially single-stranded. Aspects of the present invention in which the substrate molecule is single-stranded involve the use of a single-stranded ligase.

Some applications of the present invention involve the ligation of adapter sequences to double-stranded DNA produced by primer extension synthesis, rather than to a native or naturally double-stranded DNA substrate molecule. An example of such an application is a DNA library generated by: (a) ligation of oligonucleotides containing primer binding sequences to the 3' end of single-stranded or double-stranded DNA to allow primer extension, (b) extension of primers annealed to the oligonucleotides, and (c) ligation of 3' and 5' adapters to the ends of the double-stranded DNA produced by such primer-extension.

It is contemplated that the length of the double-stranded portion or the single-stranded portion of the substrate molecule is about 3 to about 1 x 10 ⁶ nucleotides. In some aspects, the length of the substrate molecule is about 10 to 3000 nucleotides, or about 40 to 2000 nucleotides, or about 50 to 1000 nucleotides, or about 100 to 500 nucleotides, or about 1000 to 5000 nucleotides, or about 10,000 to 50,000 nucleotides, or about 100,000 to 1 x 10 ⁶ nucleotides. In other aspects, the length of the substrate molecule is at least 3 and at most about 50, 100, or 1000 nucleotides; or at least 10 and at most about 50, 100, or 1000 nucleotides; or at least 100 and at most about 1000, 5000, or 10000 nucleotides; or at least 1000 and at most about 10000, 20000, and 50000; or at least 10000 and at most about 20000, 50000, and 100,000 nucleotides; or at least 20000 and at most about 100,000, 200,000, or 500,000 nucleotides; or at least 200,000 and at most about 500,000, 700,000, or 1,000,000 nucleotides. In various aspects, the length of the substrate molecule is about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, or about 43. , about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82 2. About 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270 , about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590 0, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 9 10, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900 900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, 10,000, 15,000, 20,000, 50,000, 1 00,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000 or more nucleotides.

Amplicon molecules

It will be understood that "amplicon" as used herein refers to a portion of a polynucleotide that has been synthesized using amplification techniques.

It is envisioned that the length of the amplicon is about 10 bp to 175 bp, wherein the desired amplicon size is significantly shorter than circulating cell-free DNA fragments (about 165 bp) and is small enough in size not to span formalin-induced cross-linked DNA from preserved samples, with an ideal length of <150 bp. It is envisioned that the amplicon can be 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69 bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79 bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109 8bp, 79bp, 80bp, 81bp, 82bp, 83bp, 84bp, 85bp, 86bp, 87bp, 88bp, 89bp, 90bp, 91bp, 92bp, 93bp, 94bp, 95bp, 96bp, 97bp, 98bp, 99bp, 100bp, 101bp, 102bp, 103bp, 104bp, 105bp, 106bp, 107bp, 108bp, 109bp, 110bp, 111bp, 112bp, 11 3bp, 114bp, 115bp, 116bp, 117bp, 118bp, 119bp, 120bp, 121bp, 122bp, 123bp, 124bp, 125bp, 126bp, 127bp, 128bp, 129bp, 130bp, 131bp, 132bp, 133bp, 134bp, 135bp, 136bp, 137bp, 138bp, 139bp, 140bp, 141bp, 142bp, 143bp, 144bp , 145bp, 146bp, 147bp, 148bp, 149bp, 150bp, 151bp, 152bp, 153bp, 154bp, 155bp, 156bp, 157bp, 158bp, 159bp, 160bp, 161bp, 162bp, 163bp, 164bp, 165bp, 166bp, 167bp, 168bp, 169bp, 170bp, 171bp, 172bp, 173bp, 174bp, 175bp or longer.

Alternatively, when high molecular weight DNA is used as input DNA for the amplification reaction, for longer reads, particularly for long read sequencing technologies that can provide reads of several kilobases (which provide haplotype information or span repeats or other difficult sequences (PacBio)), it is contemplated that the amplicon length is 150 bp to 150,000 bp or longer. It is contemplated that the amplicon length can be 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1,000 bp, 2,000 bp, 3,000 bp, 4,000 bp, 5,000 bp, 6,000 bp, 7,000 bp, 8,000 bp, 9,000 bp, 10,000 bp, or more. 0 bp, 11,000 bp, 12,000 bp, 13,000 bp, 14,000 bp, 15,000 bp, 16,000 bp, 17,000 bp, 18,000 bp, 19,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, 100,000 bp, 150,000 bp or longer.

In any of the methods described herein, it is envisioned that the target loci selected for multiplex amplification correspond to any diverse applications, including but not limited to oncology-specific targets, drug resistance-specific targets, drug metabolism and absorption targets (e.g., CYP2D6), targets for genetic diseases (e.g., cystic fibrosis CFTR gene, Lynch syndrome MLH1, MSH2, MSH6, PMS2, and EPCAM genes), targets from infectious pathogens, targets for pathogen host loci, species-specific targets, and any clinically available targets. In one aspect, the target loci are selected from a group of oncology targets including but not limited to BRAF, KRAS, EGFR, KIT, HRAS, NRAS, MET, RET, GNA11, GNAQ, NOTCH1, ALK, PIK3CA, JAK2, AKT1, DNMT3A, IDH2, ERBB2, and TP53. In another aspect, the oncology targets include 400-600 genes, including but not limited to the following gene subsets: ACURL1, AKT1, APC, APEX1, AR, ATM, ATP11B, BAP1, BCL2L1, BCL9, BIRC2, BIRC3, BRCA1, BRCA2, CCND1, CCNE1, CD274, CD44, CDH1, CDK4, CDK6, CDKN2A, CSNK2A1, DCON1D1, EGFR, ERBB2, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GAS6 , GATA3, IGF1R, IL6, KIT, KRAS, MCL1, MDM2, MET, MSH2, MYC, MYCL, MYCN, MYO18A, NF1, NF2, NKX2-1, NKX2-8, NOTCH1, PDCD1LG2, PDGFRA, PIK 3CA, PIK3R1, PNP, PPARG, PTCH1, PTEN, RB1, RPS6KB1, SMAD4, SMARCB1, SOX2, STK11, TERT, TET2, TIAF1, TP53, TSC1, TSC2, VHL, WT1, and ZNF217. In another embodiment, the target locus is selected from a subset of genes known to have clinical relevance in oncology, including but not limited to ABI1, ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, AKT1, AKT2, ALDH2, ALK, ALO17, AMER1, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, A TP2B3, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCL5, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPR 1A, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BTG1, BUB1B, C12orf9, C15orf21, C15orf55, C16orf75, C2orf44, CACNA1D, CAL R, CAMTA1, CANT1, CARD11, CARS, CASP8, CBFA2T1, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3 , CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDC73, CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C, CDKN2a(p14), CDX2, C EBPA, CEP1, CEP89, CHCHD7, CHEK2, CHIC2, CHN1, CIC, CIITA, CLIP1, CLTC, CLTCL1, CMKOR1, CNOT3, COL1A1, COL2A1, COPEB , COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CSF3R, CTNNB1, CUX1, CYLD, D10S170, DAXX, DCTN1, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, DUX4, EBF1, ECT2L, EGFR, EIF3E, EIF4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4 , EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETV1, ETV4, ETV5, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZ R, FACL6, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXO11, FBXW7, FCGR2B, FEV, FGFR1, FGFR1OP, FGFR2, FGFR3, FH, FHIT, FIP1L1, FLI1, FLJ27352, FLT3, FNBP1, FOXA1, FOXL2, FOXO1A, FOXO3A, FOXO4, FOXP1, FSTL3, FUBP1, FUS, F VT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA11, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A, H3F3B, HCMOGT-1, HEAB ,HERPUD1, HEY1, HIP1, HIST1H3B, HIST1H4I, HLA-A, HLF, HLXB9, HMGA1, HMGA2, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, HSPCA, HSPCB, IDH1, IDH2, IGH\, IGK, IGL, IKZF1, IL2, IL21R, IL6ST, IL7R, IRF4, IRTA1, ITK, JAK1, JAK2, JAK3, JAZF1, JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA1549, KIAA1598, KIF5B, KIT, KLF4, KLK2, KMT2D, KRAS, KTN1, LAF4, LASP1, LCK, LCP1, LCX, LHFP, LIFR, LMNA, LMO1, LMO2, LPP, LRIG3, LSM14A, LYL1, MAF, MAFB, MALA T1, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDS1, MDS2, MECT1, MED12, MEN1, MET, MITF, MKL1, MLF1, MLH1 , MLL, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH , MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYO5A, MYST4, NAB2, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFATC 2. NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH1, NOTCH2, NPM1, NR4A3, NRAS, NRG1, NSD1, NT5C2, NTRK1, NTRK3, NUMA1, NUP214, NUP98, NUTM2A, NUTM2B, OLIG2, OMD, P2RY8, PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, P DE4DIP, PDGFB, PDGFRA, PDGFRB, PER1, PHF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG1, PLCG1, PML, PMS1, PMS2, PMX1 , PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPFIBP1, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1A, PSIP1, PTCH1, PTEN, PTP N11, PTPRB, PTPRC, PTPRK, PWWP2A, RAB5EP, RAC1, RAD21, RAD51L1, RAF1, RALGDS, RANBP17, RAP1GDS1, RARA, RB1, RBM15, R ECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RSPO2, RSPO3, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB , SDHC, SDHD, 42253, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMAD4, SMARCA4, SM ARCB1, SMARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SS18, SS18L1, SSX1, SSX2, SSX4, STAG2, STAT3, STAT5B, STAT6, STK11, S TL, SUFU, SUZ12, SYK, TAF15, TAL1, TAL2, TBL1XR1, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFE B. TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TOP1, TP53, TPM3, TPM4, TPR, TRA, T RAF7, TRB, TRD, TRIM27, TRIM33, TRIP11, TRRAP, TSC1, TSC2, TSHR, TTL, U2AF1, UBR5, USP6, VHL, VTI1A, WAS, WHSC1, WHSC1 L1, WIF1, WRN, WT1, WWTR1, XPA, XPC, XPO1, YWHAE, ZCCHC8, ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9 and ZRSR2.

In another aspect, the target is specific for drug resistance loci, including loci that confer resistance to tyrosine kinase inhibitors used as targeted anti-tumor agents, other targeted loci for targeted anti-tumor agents, antibiotic resistance loci, and antiviral resistance loci.

In another aspect, detection can be performed for enteric, blood-borne, CNS, respiratory, sexually transmitted, and urinary tract pathogens, including bacteria, fungi, yeasts, viruses, or parasites. Detection can also be performed for pathogens that cause ear, skin, or eye infections. Differentiation between pathogenic variants of bacteria or viruses can be performed, as well as differentiation between genes that promote antibiotic resistance or encode toxins.

The types of genetic damage that can be detected by sequence analysis of the resulting amplicons include SNVs (single nucleotide variants), point mutations, transitions, transversions, nonsense mutations, missense mutations, single base insertions and deletions, larger insertions and deletions mapped between primer pairs, known chromosomal rearrangements such as translocations, gene fusions, deletions, insertions, wherein the primer pairs are designed to flank the breakpoints of the known rearrangements; copy number changes, including amplification events, deletions and loss of heterozygosity (LOH), aneuploidy, uniparental disomy, and other inherited or acquired chromosomal abnormalities. In addition, if bisulfite conversion is performed before multiplex PCR, and primers are designed for bisulfite-converted DNA and optionally do not overlap with CpG dinucleotides (which can result in different modified sequence states, making primer design more complicated), methylation changes can also be detected using the methods disclosed herein.

For amplification of the target locus, the optimal length of the 3' target-specific portion of the primer is 15-30 bases, but is not limited to this range, wherein the target-specific portion of the primer is 5-50 bases or 10-40 bases or any length therebetween. The required Tm, determined in 2.5 mM Mg2 ⁺ , 50 mM NaCl, and 0.25 μM oligonucleotide, is 63°C, with the Tm variation between multiple primers not exceeding ±2.5°C to ensure amplification even under fixed reaction conditions. The required GC content of the target-specific portion of the primer is preferably 50%, but can vary between 30% and 70%. Target-specific primers are designed to avoid overlap with repetitive, non-unique sequences or SNP polymorphisms or known mutations that are common to the conditions being tested to ensure specific, unbiased amplification from DNA samples from different genetic backgrounds. In addition, the target-specific target and complementary primer designs should not be constrained by secondary structure formation (which may reduce performance).

The universal primer comprises a cleavable base, including but not limited to deoxyuracil, deoxyinosine or RNA, and may comprise one, two, three, four, five or more cleavable bases. In addition, the target-specific primer and the universal primer comprise one, two, three, four or more nuclease-resistant moieties at their 3' ends.

adapter molecules

The present invention envisions the use of 5' adapters and 3' adapters (see Figure 3). According to the present invention, the 3' adapter is optionally double-stranded and comprises "oligonucleotide 1" and "oligonucleotide 2". For the double-stranded substrate molecule, it is envisioned that oligonucleotide 1 and oligonucleotide 2 of any length can anneal to each other under standard reaction conditions. Therefore, the complementarity between oligonucleotide 1 and oligonucleotide 2 enables them to anneal to each other. In different embodiments, the complementarity is from about 70%, 75%, 80%, 85%, 90%, 95% to about 100% or about 70%, 75%, 80%, 85%, 90%, to about 95%, or about 70%, 75%, 80%, 85% to about 90%. In a specific embodiment, the degree of complementarity between oligonucleotide 1 and oligonucleotide 2 is 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In other embodiments, oligonucleotide 2 comprises nucleotides that are susceptible to degradation/removal, such as abasic nucleotides or ribonucleotides. In certain embodiments, oligonucleotide 1 and oligonucleotide 2 have different lengths, and oligonucleotide 1 hybridizes to any position along the length of oligonucleotide 2.

In another embodiment, the 5' adapter is single-stranded. In the embodiment of the oligonucleotide 1 in which the 5' adapter is hybridized to the 3' adapter, in another embodiment, it is envisioned that the annealing causes a breach, a gap or one or more overlapping bases (see Figure 8) between the 5' adapter and the substrate molecule. In different embodiments, the quantity of the gap or overlapping bases is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 bases in length. In another embodiment in which the 3' adapter is double-stranded, after the 5' adapter is annealed to the 3' adapter, an enzyme is added to translate the catalysis 5' adapter into the 3' adapter to remove oligonucleotide 2. In some embodiments, the 5' adapter further comprises a random single, double or more N bases at its 3' end that are not complementary to oligonucleotide 1 and which can anneal to one or more first bases of the substrate molecule (if its 5' base is replaced). In other embodiments, the 5' adapter is a modified polynucleotide. Contemplated useful modified oligonucleotides are disclosed in U.S. Patent Application Publication No. 2011/0129832, which is incorporated herein by reference in its entirety. In specific embodiments, the 5' adapter comprises a base modification selected from the group consisting of locked nucleic acid (LNA) and peptide nucleic acid (PNA). In certain embodiments, the 5'-adapter oligonucleotide is pre-annealed to the 3'-adapter (see Figure 8).

The present invention also contemplates the use of a universal adapter incorporated by PCR, a single-stranded 5' adapter, and the remainder of the 3' adapter attached to one strand of the universal adapter on a partially processed amplicon substrate. According to the present invention, the attachment of the remainder of the 3' adapter is mediated by a linker. For the linker molecule, any length complementary to the remainder of the universal adapter and the 3' adapter is contemplated, as long as the three oligonucleotides are able to anneal to each other under standard reaction conditions. Thus, the complementarity enables them to anneal to each other. In various embodiments, the complementarity is from about 70%, 75%, 80%, 85%, 90%, 95% to about 100%, or about 70%, 75%, 80%, 85%, 90%, to about 95%, or about 70%, 75%, 80%, 85% to about 90%.

In another embodiment, the 5' adapter is single-stranded. In embodiments where the 5' adapter hybridizes to the 3' overhang of the universal adapter at the end of the amplicon, in another embodiment, it is envisioned that the annealing results in a gap or a gap between the 5' adapter and the amplicon substrate. In various embodiments, the length of the gap is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases.

It is contemplated that the length of the remaining portion of the universal adapter, 5' adapter B, or 3' adapter A is about 5 to about 200 nucleotides. In some aspects, the length of the universal adapter, 5' adapter, or 3' adapter is about 5 to about 200 nucleotides, or about 5 to about 150 nucleotides, or about 5 to about 100 nucleotides, or about 5 to about 50 nucleotides, or about 5 to about 25 nucleotides, or about 10 to 200 nucleotides, or about 10 to 100 nucleotides. In other aspects, the length of the 5' adapter or the 3' adapter is at least 5 and up to about 50, 100 or 200 nucleotides; or at least 10 and up to about 50, 100 or 200 nucleotides; or at least 15 and up to about 50, 100, or 200 nucleotides; or at least 20 and up to about 50, 100 or 200 nucleotides; or at least 30 and up to about 50, 100 or 200 nucleotides; or at least 40 and up to about 50, 100 or 200 nucleotides. In various aspects, the length of the substrate molecule is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75 about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 310 0, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, about 5100, about 5200, about 5300, about 5400, about 5500, about 5600, about 5700, about 5800, about 5900, about 6000, about 6100, about 6200, about 6300, about 6400, about 650 0, about 6600, about 6700, about 6800, about 6900, about 7000, about 7100, about 7200, about 7300, about 7400, about 7500, about 7600, about 7700, about 7800, about 7900, about 8000, about 8100, about 8200, about 8300, about 8400, about 8500, about 8600, about 8700, about 8800, about 8900, about 9000, about 9100, about 9200, about 9300, about 9400, about 9500, about 9600, about 9700, about 9800, about 990 0, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000 , about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, about 30,000, about 30,500, about 31,000, about 31,500, about 32,000, about 32,500, about 33,000, about 33,500, about 34,000, about 34,500, about 35,000, about 35,500, about 36,000, about 36,500, about 37,000, about 37,500, about 38,000, about 38,500, about 39,000, about 39,500, about 40,000, about 40,500, about 41,000, about 41,500, about 42,000, about 42,500, about 43,000, about 43,500, about 44,000, about 44,500, about 45,000, about 45,500, about 46,000, about 46,500, about 47,000, about 47,500, about 48,000, about 48,500, about 49,000, about 49,500, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000 or more nucleotides.

In order to complete the NGS adapter connection, the universal adapter primer also includes modified bases and/or connections that can be destroyed by enzymatic, chemical or physical means. Modifications include but are not limited to dU-bases, deoxyinosine and RNA bases. As a result of the degradation of one chain of the universal adapter corresponding to the universal primer with a cleavable base, the single-stranded 5' adapter anneals to the 3' protrusion of the amplicon. In some embodiments, the degradation is carried out enzymatically, more specifically, by using uracil-DNA glycosidase (UDG), or a combination of UDG and apurinic/apyrimidinic nuclease (if the oligonucleotide contains deoxyuracil bases), or by endonuclease V (if the oligonucleotide contains deoxyinosine bases). If the included primer contains RNA bases, degradation can also be carried out by incubation with RNase H1 or RNase H2. In some applications, the degradation of the included primer can be carried out by chemical or physical (e.g., by light). Alternatively, the 3' protrusion of the amplicon can be produced by limited exonuclease digestion of the 5' end of the amplicon. The limited digestion can be achieved enzymatically, more specifically by using T7 gene 6 exonuclease or λ5'→3' exonuclease if the primer oligonucleotide contains one or more nuclease-resistant bases at the 3' end, in particular one or more bases with phosphorothioate linkages. In this case, the exonuclease reaction stops at the modified bases and generates a 3' overhang.

Methods – Steps

The first three incubations of the method are pre-ligation steps and include (i) dephosphorylation, (ii) finishing and (iii) optional adenylation. The remaining two incubations of the method include (1) 3' adapter ligation, and (2) 5' adapter ligation, which include (a) 5' adapter annealing (b) removal of 5' bases from the substrate molecule and (c) 5' adapter ligation (see Figures 4-6). In this aspect, the method has up to 3 pre-ligation steps and 2 ligation steps. In another aspect, if the substrate molecule contains a pre-existing 3' overhang, preferably as a 3' adapter, the method has a single ligation step of a 5' adapter (see Figure 7a).

In the amplification reaction, the number of multiplex cycles is limited to a minimum of 2, or 3 cycles, 4 cycles, 5 cycles or more, up to N cycles, can be performed before switching to a non-multiplex universal adapter single primer amplification. The number of universal cycles can be from 1 cycle to 40 or more cycles, depending on the DNA input and the desired library yield. After the multiplex PCR amplification, a purification step is performed, followed by a simultaneous adapter ligation step.

Pre-connection steps:

(I) Dephosphorylation

Before the adapter is connected, the DNA ends are optionally processed to improve the efficiency of the adapter connection reaction. DNA end processing in existing methods generally uses two enzymatic reactions: (a) incubation with one or more proofreading DNA polymerases to finish the DNA ends by removing 3'-protrusions and filling the recessed 3' ends, and (b) incubation with polynucleotide kinases to add phosphate groups to the 5' ends. When processing DNA ends, some methods also adenylate the blunt-ended DNA at the 3' end by incubating the finished DNA with a non-proofreading DNA polymerase. Adenylylation helps prevent DNA self-ligation and the formation of chimeric products. It also minimizes the formation of adapter-dimers, which is attributed to the presence of dT at the 3' end of the corresponding adapter. The present invention solves these problems in a completely different way. Instead of adding phosphate groups to the 5' ends of the DNA fragments, the method of the present invention performs the optional complete removal of phosphate groups from the 5' ends of the DNA fragments. Dephosphorylation of the DNA ends is achieved by incubating the DNA fragments with an enzyme that can remove phosphates from the DNA ends. Examples of enzymes that can be used in the methods of the present invention to remove 5' or 3' phosphates include, but are not limited to, any phosphatase, such as calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, and placental alkaline phosphatase, each used according to standard conditions.

(ii) Finishing

After removing alkaline phosphatase or inactivating it by heating, the DNA substrate molecule is optionally incubated with a proofreading DNA polymerase in the presence of dNTPs to produce blunt ends. The reaction is carried out according to standard conditions. Dephosphorylated and finely processed DNA fragments are good substrates for joining 3 ' adapters, but they are poor substrates for DNA fragment concatemer connection and chimera formation. They are also poor substrates for the connection of conventional adapters.

In some applications of the present invention, 5'-end dephosphorylation by phosphatase can be omitted, but in this case, it is preferred to add an enzyme such as T4 polynucleotide kinase to the DNA polishing mixture to ensure that the phosphate group is removed from the 3' end before DNA polishing. Alternatively, the first two pre-ligation reactions described above, dephosphorylation and polishing, can be performed in any order and result in blunt-ended, double-stranded DNA lacking a 5' phosphate group at its terminus.

(iii) Adenylylation

The present invention also contemplates the use of adenylylation of the 3' termini of blunt-ended DNA fragments using DNA polymerases with non-template polymerase activity, including but not limited to the Klenow fragment of DNA polymerase I (lacking 3' exonuclease activity (exo-)), and Taq DNA polymerase. Both alkaline phosphatase treatment and adenylylation reduce the occurrence of self-ligation of DNA fragments and the formation of chimeric library molecules. In the case of including an adenylylation step, the 3' adapters used in subsequent steps will require a single T overhang.

Connection steps:

(1) 3’ adapter ligation, or generation of single-stranded 3’ overhangs on DNA substrates

The options are shown in Figure 7.

Option 1a: 3' blocked oligonucleotide 2 as part of a double-stranded 3' adapter (Figure 7a)

Existing NGS library preparation schemes rely on the connection between the 3'OH group of the adapter and the 5'phosphate group at the end of the DNA fragment. For this reason, the adapter used in conventional methods generally has a functional double-stranded end with a 3'hydroxyl group and an optional 5'phosphate group (see Figures 1 and 2). In contrast, the present invention adopts a connection reaction between the 5'phosphate group of the 3' adapter and the 3'OH group of the DNA fragment, leaving a gap between the 3' end of the 3' adapter and the 5' end of the DNA fragment (see Figure 3). The 3' adapter has a functional double-stranded end with a 5'-phosphate group, and in this option, it is not competent for the 3' nucleotides (e.g., base analogs containing sugar modifications, such as 2', 3' dideoxy bases or 3'-deoxy bases) to be connected. The 3' adapter is formed by annealing two oligonucleotides: oligonucleotide 1 having a phosphate group at the 5' end and a blocking protective group (e.g., C3 spacer) at the 3' end, and oligonucleotide 2 lacking a phosphate group at the 5' end and containing a non-connectable base at the 3' end. Oligonucleotide 2 also comprises modified bases and/or connections that can be broken by enzymatic, chemical or physical means. In most applications, the end of the 3' adapter that is connected to the substrate molecule is a blunt end. In applications involving adenylylation of DNA fragments, the connectable end of the 3' adapter has a 3' overhang that comprises 2', 3' dideoxythymine or 3'-deoxythymine bases (or other modifications of thymine bases that block its ability to form covalent bonds with adjacent bases). In other applications, the functional end of the 3' adapter may have a 3' or 5' overhang that comprises multiple bases. During the incubation process using DNA ligase, the 5' phosphate of the 3' adapter becomes connected to the 3' end of the DNA substrate molecule, leaving a gap between the 3' end of the 3' adapter and the 5' end of the DNA substrate molecule. After the reaction is complete, the connected DNA is purified by a spin column or SPRI bead-based purification method to remove excess adapters and other components of the ligation reaction.

Option 1b: 3' hydroxy oligonucleotide 2 as part of the double-stranded 3' adapter (Figure 7a)

In an alternative approach, a 3'-adapter lacking a closed, non-connectable base at the 3' end of oligonucleotide 2 is employed. The connection of the non-blocked oligonucleotide 2 to the substrate molecule will still be prevented by lacking the 5' phosphate on the substrate molecule due to the dephosphorylation reaction. The advantage of employing an unblocked oligonucleotide 2 is that the 3' end of oligonucleotide 2 can be extended by a single base using a dideoxynucleotide mixture and a DNA polymerase capable of gap-translational DNA synthesis. This enables an alternative approach to be adopted for 5' base excision of the substrate molecule, with subsequent steps being described below. The disadvantage of employing an unblocked 3'-adapter is that an adaptor-dimer is produced during the ligation reaction, which reduces adaptor concentration and may thereby reduce adaptor connection efficiency. Similarly, for this option, oligonucleotide 2 also comprises modified bases and/or connections that can be destroyed enzymatically, chemically or physically.

Option 2: Single-stranded 3’ adapter (Figure 7a)

In the presence of a ligase (DNA or RNA) capable of covalently binding a single-stranded adaptor to a double-stranded (or single-stranded) substrate molecule, oligonucleotide 2 can be omitted from the reaction.

Option 3: Homopolymer 3' adapter (Figure 7a)

In the presence of a template-independent polymerase such as terminal deoxynucleotidyl transferase (TdT), poly (A) polymerase, poly (U) polymerase, or a DNA polymerase lacking 3'-exonuclease proofreading activity, and comprising nucleotides, a homopolymer or other tail can be incorporated into the 3' end of the substrate molecule, which can serve as a 3' adapter sequence.

Option 4: Controlled tailing and simultaneous 3' adapter ligation (Figure 7a)

In the presence of a template-independent polymerase such as TdT, nucleotides, and also comprising a ligase and an attenuator-adapter molecule, the synthesized tail and the defined 3' adapter sequence can be incorporated into the 3' end of the substrate molecule. See International Patent Application No. PCT/US13/31104, filed March 13, 2013, which is incorporated herein by reference in its entirety.

Option 5: Omit the 3’ adapter ligation step (Figure 7b)

In cases where the substrate molecule contains a pre-existing 3' overhang (defined or random sequence) that occurs naturally or results from previous enzymatic or other treatments, a separate 3' adaptor ligation step is not necessary and can be omitted, wherein the pre-existing 3' overhang can serve as a 3' adaptor.

In an alternative embodiment, a phosphatase with zinc and other reaction components can be added to the 3' adapter ligation reaction at the completion of the reaction. Performing the phosphatase reaction after 3' adapter ligation is a means of rendering any unligated 3' adapter molecules unable to subsequently ligate, which prevents the formation of adapter dimers in subsequent steps when a 5' adapter is present.

(2) 5' adapter ligation, which involves three steps occurring in a single incubation

(I) Annealing of the 5' adapter

In the case of single-stranded 3’ adapter ligation (option 2), homopolymer addition (option 3), or using pre-existing 3’ overhangs as 3’ adapters (option 5), annealing of the 5’ adapter can be performed directly without further considerations since there is no oligonucleotide 2 that needs to be degraded or replaced.

When ligation of double-stranded 3'-adapters is used to generate single-stranded 3' overhangs at the ends of double-stranded DNA (Options 1a, 1b, and 4 above), any of five different options can be used to anneal the 5'-adapter to the 3'-adapter, each of which is discussed below and illustrated in Figure 8:

i) After degradation of oligonucleotide 2 annealed to the 3' adapter

ii) by competitive displacement of oligonucleotide 2 annealed to the 3' adapter

iii) by further annealing the 5' adapter to the 3' end of oligonucleotide 1 relative to the annealing site of oligonucleotide 2, followed by gap translation and degradation of oligonucleotide 2

iv) by pre-annealing the 5' adapter to the 3' region of oligonucleotide 1 of the 3' adapter, followed by gap translation and degradation of oligonucleotide 2

v) by pre-annealing a 5' adapter with a 3' blocking group to the 5' region of oligonucleotide 1 (instead of oligonucleotide 2) of the 3' adapter, followed by enzymatic cleavage of the 3' blocking group

Option i:

The oligonucleotide 2 of the 3' adapter also includes modified bases and/or connections that can be destroyed by enzymatic, chemical or physical means. Modifications include but are not limited to dU-bases, deoxyinosine and RNA bases. Annealing of the single-stranded 5' adapter to the 5' portion of the oligonucleotide 1 of the 3' adapter occurs as a result of partial degradation of the 3' adapter (partial degradation of oligonucleotide 2). In some embodiments, degradation of oligonucleotide 2 is achieved by enzymatic methods, more specifically, by using uracil-DNA glycosidase (UDG), or a combination of UDG and apurinic/apyrimidinic endonuclease (if the second oligonucleotide includes deoxyuracil bases), or by endonuclease V (if the second oligonucleotide includes deoxyinosine bases). If the second oligonucleotide includes RNA bases, degradation of oligonucleotide 2 can also be carried out by incubation with RNase H1 or RNase H2. In some applications, degradation of the second oligonucleotide can be accomplished by chemical or physical (e.g., by light) means.

Option ii:

In some applications, annealing of the 5' adapter to the oligonucleotide 1 of the 3' adapter occurs without degrading oligonucleotide 2. In this case, replacing oligonucleotide 2 with a single-stranded 5' adapter can be facilitated by utilizing the higher affinity of the 5' adapter over oligonucleotide 2, due to increased complementarity between oligonucleotide 1 and the 5' adapter sequence or due to base modifications (e.g., LNA bases) within the 5' adapter that increase its melting temperature. Depending on the design of the 5' adapter, oligonucleotide 1 annealing to the 3' adapter may result in a gap or gap between the 3' end of the 5' adapter and the 5' end of the DNA substrate molecule, or correspondingly, an overlap of the 3' and 5' bases of the 5' adapter and the DNA substrate molecule.

Option iii:

In this case, no degradable modification or competitive displacement of oligonucleotide 2 is employed. Instead, the 5' adapter displaces oligonucleotide 2 by annealing to the 3' adapter, and further, the 3' of oligonucleotide 1, relative to the annealing site of oligonucleotide 2, followed by limited nick translation "chewing forward", which results in degradation or partial degradation of oligonucleotide 2.

Options iv and v:

In these cases, the 5' adapter forms part of the 3' adapter and is present during the ligation of the 3' adapter to the DNA substrate. In option iv, the 5' adapter is pre-annealed to the 3' adapter, further to the 3' of oligonucleotide 1, relative to the annealing site of oligonucleotide 2 (similar to option iii). In option v, the 5' adapter has a blocking group at the 3' end and is pre-annealed, replacing oligonucleotide 2 with the 3' adapter. After ligation of the 3' adapter, the blocking group at the 3' end of the 5' adapter is enzymatically removed to allow its extension by a DNA polymerase.

(II) Removal of the 5'-base from the substrate molecule results in exposure of the 5' phosphate

In this step, the generation of a ligation-compatible 5' terminal phosphate group on the substrate molecule is achieved by removing the damaged 5' terminal base of the DNA substrate molecule by nick translation of the 5' adapter oligonucleotide using a DNA polymerase and nucleotides (option i), a displacement-cleavage reaction using a 5' adapter and a 5'-flanking endonuclease in the absence of nucleotides (option ii), or a single dideoxy base extension from oligonucleotide 2 followed by displacement-cleavage using a 5'-flanking endonuclease in the absence of nucleotides (option iii). For the third option, 5' base excision of the substrate molecule occurs before annealing of the 5' adapter, as it is alternatively performed using annealed oligonucleotide 2 rather than a 5' adapter, but is included in this section to simplify the description of the method (see Figure 9).

Option i:

Gap translation DNA synthesis is initiated at the gap or gap between the 3' end of the 5' adapter oligonucleotide and the 5' end of the DNA substrate molecule and stops when the ligation reaction closes the gap (see Figures 4 and 6a). The gap translation reaction can be carried out by, but is not limited to, DNA polymerases such as DNA polymerase I (holoenzyme), Taq DNA polymerase, Tth DNA polymerase, and BstDNA polymerase (holoenzyme). Other enzymes envisioned for use include, but are not limited to, DNA polymerases with 5'-3' exonuclease activity, 5' flanking endonucleases, and combinations of strand displacement polymerases and 5' flanking endonucleases.

Contemplated reaction conditions for this step include those in which (i) both a polymerase with endogenous 5' exonuclease activity and a ligase are active; (ii) both a strand-displacing polymerase and a flanking endonuclease polymerase and a ligase are active; (iii) both a flanking endonuclease and a ligase are active, (iv) both a thermostable enzyme and a thermolabile enzyme are active; or (v) only thermostable or only thermolabile enzyme activity can occur. In some embodiments, conditions (i) and (ii) are each performed using dNTPs for nick translation. In a specific embodiment, Taq polymerase and E. coli ligase are used at a reaction temperature of 40°C. However, in various embodiments, a reaction temperature range of 10°C to 75°C is contemplated.

Before ligation occurs between the 5' adapter extension product and the DNA substrate molecule, the gap translation reaction causes one, two or more bases to be removed from the 5' end of the DNA substrate molecule. Gap translation synthesis can occur in the presence of all four nucleotides dGTP, dCTP, dTTP and dATP or their restricted combinations. Restricted combinations include but are not limited to tri-nucleotide combinations, such as dGTP, dCTP and dATP, or dGTP, dCTP and dTTP, or dGTP, dATP and dTTP, or dCTP, dATP and dTTP, di-nucleotide combinations, such as dGTP and dCTP, or dGTP and dATP, or dGTP and dTTP, or dCTP and dATP, or dCTP and dTTP, or dATP and dTTP, or only mononucleotides, such as dGTP, or dCTP, or dATP, or dTTP.

Option ii:

The displacement-cleavage reaction does not require dNTPs but requires that the 5' adapter sequence contains one, two or more random bases at the 3' end to overlap with the substrate molecule, and that multiple 5' adapters are included in the reaction (see Figures 5 and 6b). The displacement-cleavage reaction is initiated by annealing of the 5' adapter, displacement of the 5' DNA base of the DNA substrate molecule that overlaps with the 3' base of the 5' adapter, and cleavage of the replaced base by a 5'-flank endonuclease. In some embodiments, the 5' adapter has a random base dN at the 3' end. In this case, the overlap involves one base and only a single 5' base will be removed from the 5' end of the DNA substrate molecule and replaced with a similar base from the 5' adapter sequence. The efficiency of the displacement-cleavage reaction is increased by cycling the reaction temperature from 40°C to 65°C to allow the 5' adapter to dissociate and reanneal (if its terminal 3' base is mismatched to the 5' base of the DNA substrate molecule).

Option iii:

In the previous step, an alternative embodiment of the 5' adapter participating in the 5' base excision of the substrate molecule would replace oligonucleotide 2 that makes the 3' adapter participate in the 5' base excision of the substrate molecule (see Figure 9).

In one approach (Fig. 9a and c), the oligonucleotide 2 of the 3' adapter comprises an extendable 3' end, and in the presence of a dideoxynucleotide mixture and a polymerase, under suitable conditions, a single dideoxy base addition occurs, which results in a single base overlap with the 5' end of the substrate molecule, which induces a single base replacement-cleavage performed by a suitable flanking endonuclease or a polymerase with 5' flanking endonuclease activity. Subsequently, a 5' adapter (Fig. 9a) having random dN bases at its 3' end is employed, wherein a gap is formed after binding to the 3'-adapter, which is joined to the end of the double-stranded DNA. The gap can be sealed by a DNA ligase, which results in the covalent attachment of the 5' adapter to the 5' end of the DNA substrate molecule.

Alternatively, a 5' adapter oligonucleotide lacking a random dN base at its 3' end can be used (Figure 9c), which forms a single base gap after binding to the 3'-adapter, which is joined to the end of the double-stranded DNA substrate molecule. This gap can be filled by a DNA polymerase lacking strand-displacement activity (e.g., T7 or T4 DNA polymerase) to create a gap, which can then be closed by DNA ligase, resulting in covalent attachment of the 5' adapter to the 5' end of the DNA substrate molecule.

In another alternative scenario (see Figures 9b and d), oligonucleotide 2 comprising a blocked 3' end is partially degraded or displaced by a primer oligonucleotide that is gradually extended with a single dideoxy-base by a DNA polymerase having 5' flanking endonuclease activity, resulting in the excision of a single base from the 5' end of the DNA. The primer oligonucleotide, in turn, is gradually degraded or replaced by a 5' adapter having a random dN base at its 3' end to create a gap that can be closed by a DNA ligase.

(III) Ligation of 5' adapter

The covalent attachment of the 5' adapter to the substrate molecule involves the connection between the 5' adapter or its extension product and the exposed 5' phosphate of the substrate molecule. When the excision of one or more 5' bases of the DNA substrate molecule is achieved by a nick translation reaction, the ligation reaction closes the gap between the polymerase-extended 5' adapter and the cut 5' end of the DNA substrate molecule. When the excision of the 5' base of the DNA substrate molecule is achieved by a displacement-cleavage reaction, the connection occurs between the original 5' adapter oligonucleotide and the cut 5' end of the DNA substrate molecule. In different embodiments, the standard conditions for the ligation reaction in this step include the use of any DNA ligase that can close the gap or gap in the DNA. In one embodiment, the ligase is Escherichia coli DNA ligase, and the reaction occurs in a temperature range of 10°C to 50°C. In some embodiments, the ligase is a thermostable DNA ligase, for example, Taq DNA ligase, or Ampl ligase, and the reaction occurs in a temperature range of 30°C to 75°C.

In various aspects of the invention, the three steps (I), (II), and (III) of the 5' adapter ligation step are performed simultaneously in a single incubation by mixing and incubating the 3'-adapted substrate DNA and (i) an optional degrading endonuclease (e.g., UDG, endonuclease V, RNase H, or a combination thereof); (ii) a nick-translating DNA polymerase or a 5'-flanking endonuclease; and (iii) a DNA ligase (see FIG6 ). The incubation is performed at a constant temperature or using temperature cycling conditions ranging from 10° C. to 75° C. In other applications, the degradation of the 3' adapter portion is performed separately from the downstream reactions.

NGS library construction

The synthesis of Illumina NGS libraries can be carried out using the methods described herein. As shown in Figure 10, Illumina libraries can be constructed using a gap translation connection method (left side) or a displacement cutting connection method (right side). The order of joining of the two Illumina adapters is flexible. In Figure 10a, Illumina adapter P7 is a 3' adapter and Illumina adapter P5 is a 5' adapter, while in Figure 10b, Illumina adapter P5 is a 3' adapter and Illumina adapter P7 is a 5' adapter. The library shown in Figure 10 can be constructed without PCR or can be constructed by PCR amplification, depending on the amount of input substrate DNA. Alternatively, the synthesis of Illumina NGS libraries can be carried out using methods disclosed herein, wherein PCR amplification is required because the method uses truncated adapter sequences (see Figure 11). In this case, P5 or P7 is introduced as a truncated adapter (only P7 is shown), and, following amplification using PCR primers that introduce the full-length adapter sequence and contain a degradable base at its 5' end, followed by degradation of the 5' portion of the resulting amplicon, P7 or P5 can be introduced by annealing and ligation. If, instead, a truncated degradable primer is used for PCR amplification, a bridge-ligation of the remaining portion of the adapter can be performed to complete the full-length sequence.

The methods disclosed herein can be used to construct NGS libraries for a variety of sequencing platforms, and another example is shown in Figures 12 and 13, which describe ion torrent library construction. As shown in Figure 12, by introducing a partial duplication of the A adapter sequence on the P1 adapter at the insertion junction, subsequent annealing of the 5' adapter after the 3' adapter connection can occur. The order of connection is flexible, wherein the adapter P1, which has a partial duplication with the adapter A, can be introduced as the 3' adapter, and then the adapter A as the 5' adapter is connected using gap translation or displacement cutting (Figure 12a). Alternatively, the adapter A can be introduced as the 3' adapter, and the adapter P1, which has a partial duplication with the adapter A, can be the 5' adapter (Figure 12b). Because the ion torrent sequencing is performed as a single read from the A adapter, due to the length of the partial duplication of the adapter A on the P1 adapter, it will not interfere with the sequencing primer annealing or other adapter functions.

Alternatively, in Figure 13, combinatorial barcodes can be introduced into an Ion Torrent library using the described method. During the 3' adapter ligation step, the first part of the dual combinatorial barcode is introduced adjacent to the linker region L common to all 20 barcodes. After degradation of the 3' closed strand that is not attached to the DNA substrate, the 5' adapter is annealed to the common linker region L, which introduces the second part of the dual barcode 5' adjacent to the linker region L. After gap translation ligation, the resulting library can be amplified using standard Ion Torrent PCR primers, and when the library molecules are sequenced from the A adapter side, the sample identification of each Ion Torrent will be read at the beginning of the read, where 96 possible combinations are available.

Applications of Targeted NGS Libraries

The methods disclosed herein can be used to construct NGS libraries in which specific targets can be selected and enriched as a means of reducing complexity and sequencing requirements relative to whole genome sequencing. An example of such an application would be the joining of 3' adapters to 5' adapters to a randomly fragmented, denatured, and primer-extended DNA substrate, wherein a primer or primers are annealed to a known target DNA region. In this case, only the loci of the target will contain double-stranded ends, wherein the unselected loci will remain single-stranded, and adapter ligation will not occur on their ends.

In other applications, the 5' adapter of the present invention can be used to select and enrich a small portion of DNA fragments with known end sequences. The pre-selected DNA sequence can contain one, two, three or more terminal DNA bases. In order to achieve the selection, the 5' adapter sequence should contain a selected invading base or base combination at the 3' end. Thus, only DNA fragments with the selected end sequence will be connected to the 5' adapter and amplified. As shown in Figure 14, 5' adapters with 3' ends complementary to the end sequences of the selected restriction fragments can be used to select restriction fragment targets from a variety of restriction fragments. In another embodiment, 5' adapters with 3' ends containing CpG dinucleotides will be enriched for fragments derived from CpG islands.

Alternatively, target selection can be performed after library construction using the methods described herein (see Figure 15). If a library is constructed in which one of the adapters contains a degradable base at its 5' end, followed by target-specific primer extension and partial digestion of the degradable portion of the adapter, a biotinylated 5' adapter can be annealed to the resulting 3' overhang and covalently joined to the target DNA substrate using either nick translation ligation (Figure 15a) or displacement cleavage ligation (Figure 15b), and can then be captured using streptavidin magnetic beads and then PCR amplified to generate sufficient material for sequencing.

Design and application of alternative adapters

Several alternative adapter designs and ligation methods using the methods of the present invention are also presented. In Figure 16, a single adapter sequence is used to construct the library rather than an adapter sequence pair. In this example, the same steps are used for substrate processing followed by ligation, as well as 3' adapter ligation and 5' adapter gap translation ligation or displacement cleavage ligation, and the resulting library can be amplified by PCR using a single primer.

In Figure 17, a method for ligating a single oligonucleotide hairpin adapter is presented, wherein the 5' end of the hairpin adapter is used to perform a 3' adapter ligation to a substrate molecule, followed by degradation of the closed 3' end of the hairpin adapter, and the truncated 3' end of the hairpin adapter is used for gap translation ligation to the exposed 5' phosphate of the substrate molecule.

Sometimes, it is advantageous to produce a circular DNA library, for example, for constructing an intermediate structure of a double-ended sequencing (mate-pair) NGS library. As shown in Figure 18, the library can be constructed using the method of the present invention. In a first step, 3' adapters are connected using mutually complementary adapters X and X'. After degradation of the non-connected chain, non-covalent DNA cyclization can be carried out by the complementarity of the 3' overhangs X and X' on each substrate molecule. In order to facilitate unimolecular annealing and reduce concatemer formation, the annealing reaction is carried out with a suitable low DNA concentration. After 3' overhang annealing, gap translation connection can be carried out.

enzymes

Ligase enzymes that can be used to practice the methods of the present invention under standard reaction conditions include, but are not limited to, T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, or T7 DNA ligase, Taq DNA ligase, Amp ligase, E. coli DNA ligase, and E. coli RNA ligase. In various embodiments, the present invention contemplates reaction conditions suitable for blunt-end or cohesive ("sticky") end ligation. In some embodiments, the sticky ends comprise 5' overhangs or 3' overhangs.

Examples of enzymes that can be used in the methods of the present invention to remove 5' or 3' phosphates include, but are not limited to, any phosphatase, such as calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, and placental alkaline phosphatase, each of which is used according to standard conditions. In addition, the phosphatase activity of T4 polynucleotide kinase can be used to remove 3' phosphate groups.

Polymerases useful in practicing the present invention include, but are not limited to, DNA polymerases (which may include thermostable DNA polymerases, e.g., Taq DNA polymerase), RNA polymerases, DNA polymerase I, and reverse transcriptases. Non-limiting examples of enzymes useful in practicing the present invention include, but are not limited to, KAPA High Fidelity and KAPA High Fidelity Uracil+, VeraSeq Ultra DNA Polymerase, VeraSeq 2.0 High Fidelity DNA Polymerase, Takara PrimeSTAR DNA Polymerase, Agilent Pfu Turbo CX Polymerase, Phusion U DNA Polymerase, Deep VentR ^™ DNA Polymerase, LongAmp ^™ Taq DNA Polymerase, Phusion ^™ High Fidelity DNA Polymerase, Phusion ^™ Hot Start High Fidelity DNA Polymerase, Kapa High Fidelity DNA Polymerase, Q5 High Fidelity DNA Polymerase, Platinum Pfx High Fidelity Polymerase, Pfu High Fidelity DNA Polymerase, Pfu Ultra High-Fidelity DNA Polymerase, KOD High-Fidelity DNA Polymerase, iProof High-Fidelity Polymerase, High-Fidelity 2 DNA Polymerase, Velocity High-Fidelity DNA Polymerase, ProofStart High-Fidelity DNA Polymerase, Tigo High-Fidelity DNA Polymerase, Accuzyme High-Fidelity DNA Polymerase, DNA Polymerase, DyNAzyme ^™ II Hot-Start DNA Polymerase, Phire ^™ Hot-Start DNA Polymerase, Phusion ^™ Hot-Start High-Fidelity DNA Polymerase, Crimson LongAmp ^™ Taq DNA Polymerase, DyNAzyme ^™ EXT DNA Polymerase, LongAmp ^™ Taq DNA Polymerase, Phusion ^™ High-Fidelity DNA Polymerase, Taq DNA Polymerase with Standard Taq (Mg-Free) Buffer, Taq DNA Polymerase with Standard Taq Buffer, Taq DNA Polymerase with ThermoPol II (Mg-Free) Buffer, Taq DNA Polymerase with ThermoPol Buffer, Crimson Taq ^™ DNA polymerase, Crimson Taq ^™ DNA Polymerase with (Mg-free) buffer, Phire ^™ Hot Start DNA Polymerase, (exo-)DNA Polymerase, Hemo KlenTaq ^™ , Deep VentR ^™ (exo-)DNA Polymerase, Deep VentR ^™ DNA Polymerase, DyNAzyme ^™ EXT DNA Polymerase, Hemo KlenTaq ^™ , LongAmp ^™ Taq DNA Polymerase, AMV First Strand cDNA Synthesis Kit, M-MuLV First Strand cDNA Synthesis Kit, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, 9°Nm DNA Polymerase, DyNAzyme ^™ II Hot Start DNA Polymerase, HemoKlenTaq ^™ , Sulfolobus DNA Polymerase IV, Therminator ^™ γ DNA Polymerase, Therminator ^™ DNA Polymerase, Therminator ^™ II DNA Polymerase, Therminator ^™ III DNA Polymerase, Bsu DNA polymerase, large fragment, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment, Klenow fragment (3′→5′ exo–), phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase (unmodified), terminal transferase, reverse transcriptase and RNA polymerase, E. coli poly (A) polymerase, AMV reverse transcriptase, M-MuLV reverse transcriptase, phi6 RNA polymerase (RdRP), poly (U) polymerase, SP6 RNA polymerase, and T7 RNA polymerase.

Enzymes having flanking endonuclease activity that can be used in the present invention include, but are not limited to, flanking endonuclease 1 (FEN1), T5 exonuclease, Taq DNA polymerase, Bst polymerase, Tth polymerase, DNA polymerase I, and derivatives thereof.

Example

Example 1

Comparison of conventional adapter ligation and 3' adapter ligation using FAM-labeled oligonucleotides

Principle: Using a FAM-labeled oligonucleotide system, blunt-end ligation with either a filler adapter ( FIG. 2A ) or a 3′ adapter ( FIG. 3 ) was tested at varying substrate:adapter molar ratios to determine the effect on ligation efficiency and chimera formation.

Material:

Filler adapter contains oligonucleotides 12-900 and 13-426 (Table 1)

3' adapter; first oligonucleotide 13-340 (Table 1)

3' adapter; 2nd oligonucleotide option 1 (with blocking 3' deoxythymidine base at 3' end) 13-559 (Table 1)

3' adapter; 2nd oligonucleotide option 2 (with phosphate group at 3' end) 13-558 (Table 1)

FAM substrate A contains oligonucleotides 13-562 and 13-563, where the FAM group labels the linkage to the 5' phosphate of the substrate (Table 1)

FAM substrate B comprises oligonucleotides 13-561 and 13-564, wherein a FAM group labels the linkage to the 3' OH of the substrate, and wherein the corresponding 5' end of the substrate has a phosphate (Table 1)

FAM substrate C comprises oligonucleotides 13-560 and 13-564, wherein the FAM group labels the linkage to the 3' OH of the substrate, and wherein the corresponding 5' end of the substrate lacks a phosphate (Table 1)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

method:

Conventional adapter ligation reactions were assembled in a total volume of 10 μl containing 1× T4 DNA ligase buffer, 10 pmoles of FAM substrate A, 20 or 200 pmoles of filler adapter, 600 units of T4 DNA ligase (fast), or no ligase.

3' adapter ligation reactions were assembled in a total volume of 10 μl containing 1x T4 DNA ligase buffer, 10 pmoles of FAM substrate B or 10 pmoles of FAM substrate C, 20 or 200 pmoles of 3' adapter option 1 or 20 or 200 pmoles of 3' adapter option 2, and 600 units of T4 DNA ligase (fast) or no T4 DNA ligase.

All ligation reactions were carried out at 25°C for 30 minutes. The total ligation reaction volume (10 μl) was mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, observed on a Dark Read light box (Clare Chemical Research, catalog number S11494) and photographed with a digital camera. Subsequently, the gel was stained with gold nucleic acid gel stain (Invitrogen, catalog number S11494) (not shown).

result:

In the presence of filler adapters and T4 DNA ligase, FAM substrate A is converted into ligation products (Figure 19, lanes 1-2). This conventional adapter ligation shows that some FAM substrate A chimeras are formed when only a 2: 1 adapter: substrate ratio (Figure 19, lane 1) is used compared to a 20: 1 ratio (lane 2). In the absence of T4 DNA ligase, no ligation products are formed (Figure 19, lane 3).

Different situations of 3' adapter ligation were tested in lanes 4-12 (Figure 19). Lanes 4 and 5 show the ligation reaction between FAM substrate B and 3' adapter option 1. When using a 2:1 (lane 4) or 20:1 (lane 5) adapter: substrate ratio, a higher molecular weight chimeric product is formed, which may or may not involve a 3' adapter. However, at a 20:1 adapter: substrate ratio (lane 5), the abundance of the ligation product is higher and its formation is more favorable. Lanes 6 and 7 show the ligation reaction between FAM substrate C and 3' adapter option 1. The reaction is favorable at a 20:1 adapter: substrate ratio (lane 7) and no chimeric product is observed. Lanes 8 and 9 show the ligation reaction between FAM substrate B and 3' adapter option 2. No ligation product is observed, however, a chimeric product is detected. Lanes 10 and 11 show the ligation reaction between FAM substrate C and 3' adapter option 2. No ligation product is observed. In the absence of T4 DNA ligase, no ligation product was observed (lane 12).

in conclusion:

Conventional adapter ligation requires a 5'-phosphate on the FAM substrate, which results in the formation of chimeras if the adapter is not filled in excess. When the FAM substrate has a 5' hydroxyl group and the 3' adapter has a closed 3-deoxythymidine base (option 1), it prevents the connection between the adapter molecules and facilitates the connection between the substrate and the adapter. The connection of the 3' adapter is more efficient and almost chimera-free. In both cases, a ratio of 20:1 between the adapter and the substrate favors the formation of ligation products.

Example 2

Comparison of conventional adapter ligation and 3’ adapter ligation using sheared, size-selected genomic DNA

principle

This experiment was performed to test the effect of polishing of physically sheared genomic DNA on the efficiency of conventional or 3' adapter ligation.

Material:

Filler adapter contains oligonucleotides 13-489 and 13-426 (Table 1)

3' adapter; first oligonucleotide 13-340 (Table 1) and second oligonucleotide option 1 (containing a blocking 3' deoxythymidine base at the 3' end) 13-559 (Table 1)

NE buffer 2 (New England BioLabs, catalog number B7002S)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Adenosine 5′-triphosphate (ATP) (New England BioLabs, catalog number P0756S)

DNA polymerase I, large (Klenow) fragment (New England BioLabs, catalog number M0210S)

T4 DNA polymerase (New England BioLabs, catalog number M0203S)

T4 polynucleotide kinase (New England BioLabs, catalog number M0201S)

Exonuclease III (E. coli) (New England BioLabs, catalog number M0293S)

Antarctic phosphatase (New England BioLabs, catalog number M0289S)

Antarctic phosphatase reaction buffer (New England BioLabs, catalog number B0289S)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

Escherichia coli ATCC 11303 genomic DNA (Affymetrix, catalog number 14380)

M220 focused ultrasound system (Covaris, catalog number PN 500295)

Pippin Prep (Sage Science)

CDF2010 2% agarose, stain-free with internal standard (Sage Science)

DNA Cleaner and Concentrator-5 (Zymo Research, catalog number D4004)

25 bp ladder DNA size marker (Invitrogen (Life technologies), catalog number 10488-022)

method:

Escherichia coli genomic DNA (gDNA) was resuspended in DNA suspension buffer (Teknova, catalog number T0227) at a concentration of 100 ng/ul. The DNA was fragmented using an M220 focused ultrasonicator to an average size of 150 base pairs. Subsequently, a tight size distribution of the fragmented DNA, ranging from approximately 150 bp to approximately 185 bp, was separated on a 2% agarose gel using a Pippin Prep instrument.

The size-selected DNA of 200ng experiences the activity of different enzymes.Reaction is assembled in the cumulative volume of 30 μ l, and its comprising final concentration is 1x NE buffer 2, each dNTP of 100 μ M, 3 units of T4 DNA polymerase or 5 units of DNA polymerase I, large (Klenow) fragment or 3 units of T4 DNA polymerase and 5 units of DNA polymerase I, large (Klenow) fragment or 3 units of T4 DNA polymerase and 5 units of DNA polymerase I, large (Klenow) fragment and 1 unit of exonuclease III.Another reaction is assembled in the cumulative volume of 30 μ l, and it comprises final concentration 1x NE buffer 2,1mM ATP, 10 units of T4 polynucleotide kinase.Another reaction is assembled in the cumulative volume of 30 μ l, and it comprises final concentration 1x Antarctic phosphatase reaction buffer and 5 units of Antarctic phosphatase.Control reaction adopts 200ng of size-selected DNA and 1x NE buffer 2 to assemble. All reactions were incubated at 37°C for 30 minutes, and the DNA was purified using a DNA clean and concentrate-5 column. The DNA was eluted in 30 μl of DNA suspension buffer and divided into 2 tubes of 15 μl for subsequent conventional adapter ligation or 3' adapter ligation. Conventional adapter ligation was assembled in a total volume of 30 μl, which contained 1x T4 DNA ligase buffer, and the filler adapter contained oligonucleotides 13-489 (220 pmoles) and 13-426 (440 pmoles), and 1200 units of T4 DNA ligase (fast). The 3' adapter ligation reaction was assembled in a total volume of 30 μl, which contained 1x T4 DNA ligase buffer, 220 pmoles of 3' adapter first oligonucleotide, 440 pmoles of 3' adapter second oligonucleotide, and 1200 units of T4 DNA ligase (fast). All reactions were purified using DNA clean and concentrate-5-column. The DNA was resuspended in 10 μl of DNA suspension buffer and mixed with 10 μl of 2× formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue, and 0.01% xylene cyanol), heated at 95° C. for 5 minutes, and then run on a 6% polyacrylamide gel TBE-Urea (Invitrogen, catalog number S11494) precast in a 65° C. oven. The gel was stained with gold nucleic acid gel stain (Invitrogen, catalog number S11494) and viewed in a Dark Read light box (Clare Chemical Research) and photographed using a digital camera.

result:

Conventional adapter ligation reactions for 5' phosphates on DNA substrates that require shearing (Figure 20, top panel) show lower efficiency than 3' adapter ligations (which do not require) (Figure 20, bottom panel). After treating the DNA with only T4 DNA polymerase (lane 3) or in combination with Klenow (lane 7) or Klenow plus exonuclease III (lane 8), ligation reactions were more efficient for both types of connections. Compared to untreated DNA (lane 2), only Klenow, T4 polynucleotide kinase, or Antarctic phosphatase treatment (lanes 4, 5, and 6, respectively) resulted in only moderately enhanced blunt-end ligations. DNA fragments with tight range distribution were loaded into lane 9.

in conclusion:

The connection of blunt adapters to sheared DNA is highly dependent on the finishing of the DNA. DNA polymerases such as T4 DNA polymerase with strong 5'-3' exonuclease activity and 5'-3' polymerase activity are well suited for this purpose. Conventional adapter ligation reactions rely on the presence of intact 5' phosphates on the blunt end of the substrate. However, this is not the case with the connection of 3' adapters, because the connection occurs on the 3' hydroxyl end of the fragmented DNA. Because the 5' end of the sheared DNA is not an enzymatic substrate for T4 DNA polymerase, this explains why 3' adapters are more successfully connected than filler adapters (lane 3). The combination of T4 DNA polymerase plus Klenow and exonuclease III significantly enhances blunt end connection. Exonuclease III activity produces the blunt end required for the connection of blunt adapters by removing the 3' hydroxyl end, which can be destroyed at the 3' end of the DNA. Exonuclease III also has 3' phosphatase activity, which makes the 3' end accessible to DNA polymerase finishing activity.

Example 3

Temperature optimization for 5' adapter ligation using FAM-labeled oligonucleotide substrates

Principle: This experiment evaluates the temperature dependence and dNTP composition for nick translation-mediated 5' adapter ligation.

Material:

5' adapter oligonucleotide for gap translation (13-144) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Escherichia coli DNA ligase (New England BioLabs, catalog number M0205S)

10X E. coli DNA ligase reaction buffer (New England BioLabs)

Taq DNA polymerase, concentrated 25 U/ul (Genscript, catalog number E00012)

25 bp ladder DNA size marker (Invitrogen (Life technologies), catalog number 10488-022)

method:

The first set of nick translation reactions was assembled in a total volume of 30 μl containing a final concentration of 1× E. coli DNA ligase buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide for nick translation and 45 pmoles of oligonucleotide template, 200 μM dTTP or 200 μM of each dTTP/dGTP mixture or 200 μM of each dATP/dTTP/dGTP, and 2.5 units of Taq DNA polymerase or Taq-free DNA polymerase. The reactions were incubated at 30°C, 40°C, or 50°C for 30 minutes.

A second set of nick translation reactions after ligation was assembled in 30 ul containing a final concentration of 1x E. coli DNA ligase buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5' adapter oligonucleotide for nick translation and 45 pmoles of oligonucleotide template, 200 uM of each dATP/dTTP/dGTP, and 2.5 units of Taq DNA polymerase. Reactions were incubated at 50°C, 53°C, 56°C, or 60°C for 30 minutes. 10 μl of those reactions were used for gel analysis. 10 units of E. coli ligase were added to 20 μl, allowed to stand, and incubated at 25°C for 15 minutes. Additional control reactions were assembled in 30 ul containing a final concentration of 1x E. coli DNA ligase buffer and 30 pmoles of FAM oligonucleotide substrate. 10 μl of those reactions were mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, visualized on a Dark Read light box (Clare Chemical Research) and photographed using a digital camera.

result:

As shown in Figure 21, Figure A, Taq archaeal dna polymerase has extended the 3 ' hydroxyl end of 5 ' adapter oligonucleotide and is used for gap translation, and has removed the nucleotide on FAM oligonucleotide substrate by its 5 ' flank endonuclease activity.Only add dTTP (Figure 21, swimming lane 2,5,8, Figure A) and only allow to add a base to 3 ' end of 5 ' adapter oligonucleotide and be used for gap translation, add dTTP/dGTP (Figure 21, swimming lane 3,6,9, Figure A) and allow to add three bases, and add dTTP/dGTP/dATP (Figure 21, swimming lane 4,7,10, Figure A) and allow to add four bases, it is proportional (Figure 21, Figure A) with the base quantity cut from FAM oligonucleotide substrate.The base quantity cut from FAM oligonucleotide substrate also depends on the temperature that reaction occurs. 21, swimming lanes 5, 8, Figure A), do not allow any cutting to FAM oligonucleotide substrate, as observed at 50 ℃ (Figure 21, swimming lanes 2, Figure A).Adding dTTP/dGTP or dTTP/dGTP/dATP allows some cuttings at 40 ℃ (swimming lanes 6 and 7) or 30 ℃ (swimming lanes 9 and 10), and its efficiency is lower than 50 ℃ (swimming lanes 3 and 4).Swimming lane 1 (Figure 21, Figure A) is presented at the FAM oligonucleotide substrate in the absence of TaqDNA polymerase.

The efficiency of gap translation and the amount of the FAM oligonucleotide substrate of cutting are highly dependent on the reaction temperature. At 60 ℃, the FAM oligonucleotide substrate is almost completely processed into smaller substances (Figure 21, lane 4, Figure B). The size of the FAM oligonucleotide substrate cleavage product also decreases with the increase of reaction temperature (Figure 21, lanes 1-4, Figure B). Lane 5 (Figure 21, Figure B) is presented at the FAM oligonucleotide substrate in the absence of Taq DNA polymerase. During the gap translation reaction, Taq DNA polymerase cuts the 5' end of the FAM oligonucleotide substrate and produces terminal 5' phosphate, which is necessary for the E. coli ligase to covalently connect the 3' end of the 5' adapter oligonucleotide to the 5' end of the FAM oligonucleotide substrate. The connection efficiency depends on the temperature at which the reaction occurs. The connection product is higher in abundance at 50 ℃ (lane 6), and almost does not exist at 60 ℃ (lane 9), and a moderate amount of the connection product is produced at 53 ℃ and 56 ℃.

in conclusion:

In the gap translation process, the number of bases cut from the FAM oligonucleotide substrate depends on the complementary dNTP introduced into the reaction and the temperature at which the reaction occurs. In the process of the gap translation reaction, Taq DNA polymerase cuts the 5' end of the FAM oligonucleotide substrate and produces the terminal 5' phosphate necessary for the E. coli ligase to connect the two fragments. The FAM oligonucleotide substrate cut by gap translation at higher temperatures is a poor substrate for the connection by E. coli ligase because a gap may form between the 3' end of the 5' adapter oligonucleotide and the 5' end of the FAM oligonucleotide substrate.

Example 4

Analysis of the effect of dNTP composition on 5' adapter ligation

Rationale: This experiment was performed to assess the extent of nick translation that occurs in the presence of different dNTP compositions and the effect on paired ligation reactions.

Material:

5' adapter oligonucleotide for gap translation (13-144) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

25 bp ladder DNA size marker (Invitrogen (Life technologies), catalog number 10488-022)

Escherichia coli DNA ligase (Enzymatics, catalog number L6090L)

10X E. coli DNA ligase buffer (Enzymatics, catalog number B6090)

Taq-B DNA polymerase (Enzymatics, catalog number P7250L)

method:

Reactions were assembled in a total volume of 30 μl containing a final concentration of 1× E. coli DNA ligase buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide for nick translation and 45 pmoles of oligonucleotide template, 200 μM of each of the four dNTPs or a 200 μM mixture of the following: dCTP, dTTP, dGTP, or dATP, dTTP, dGTP, or dATP, dCTP, dGTP, or dATP, dTTP, dCTP, or no dNTPs, 10 units of E. coli ligase, and 10 units of Taq-B DNA polymerase. All reactions were incubated at 40°C for 30 minutes. Ten μl of those reactions were mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue, and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, visualized on a dark reading light box (Clare Chemical Research), and photographed with a digital camera (lower panel). The gel was then stained with gold nucleic acid gel stain (Invitrogen, catalog number S11494), visualized on a dark reading light box (Clare Chemical Research), and photographed with a digital camera (upper panel).

result:

The first two swimming lanes of Figure 22 show control oligonucleotides.In the absence of Taq-B archaeal dna polymerase, only E. coli ligase cannot connect 5 ' adapter oligonucleotide to FAM oligonucleotide substrate, because FAM substrate lacks 5 ' phosphate modification (Figure 22, swimming lane 3).In the presence of Taq-B archaeal dna polymerase and 4 kinds of dNTPs, 5 ' adapter oligonucleotide is extended to form a new product of 58 bases, and FAM oligonucleotide substrate is displaced and degraded (Figure 22, swimming lane 4) by the 5 ' flank endonuclease activity of Taq-B archaeal dna polymerase.In the presence of E. coli ligase, Taq-B archaeal dna polymerase and dATP/dTTP/dGTP (Figure 22, swimming lane 7) or dCTP/dTTP/dGTP (Figure 22, swimming lane 6) or dATP/dTTP/dCTP (Figure 22, swimming lane 9), gap translation is limited to the interpolation of four, three or one base respectively. As the 5' adapter is extended, a flank is formed at the 5' end of the FAM oligonucleotide substrate. The flank becomes a substrate for the activity of Taq-B 5' flank endonuclease, which produces the 5' phosphate required for connection. The 5' adapter is connected to the FAM oligonucleotide substrate to form a product of 69 bases. Compared to the flank of one base (Figure 22, lane 9), the flank of three or four bases (Figure 22, lanes 6 and 7) supports more efficient connection. In the presence of E. coli ligase, Taq-B DNA polymerase and dATP/dCTP/dGTP (Figure 22, lane 8), a lighter band corresponding to the connection product was observed. The weak connection activity may come from the inclusion of "unmatched" bases (A, C or G instead of T), resulting in the formation of flanks on some FAM oligonucleotide substrates. In the presence of E. coli ligase, Taq-B DNA polymerase and without dNTP, no connection product was observed. In the presence of E. coli ligase, Taq-B DNA polymerase and 4 kinds of dNTPs, the 5' adapter is connected to the FAM oligonucleotide substrate to form a 69-base product (Figure 22, lane 5). Because the 5' adapter and oligonucleotide template are excessive relative to the FAM oligonucleotide substrate, a gap translation product is also observed at 58 bases (Figure 22, lane 5, upper figure). However, the same amount of ligation product is observed. The 25bp ladder DNA size marker is loaded in lane M.

in conclusion:

The phosphorylation of 5 ' end of FAM oligonucleotide substrate is required for connection.In the presence of dNTP, need the polymerase activity of Taq archaeal dna polymerase to form the extension of 5 ' adapter, it produces flank at 5 ' end of FAM oligonucleotide substrate.This flank is good substrate for 5 ' flank endonuclease activity of Taq archaeal dna polymerase, and it produces perfect 5 ' phosphate substrate for the connection by escherichia coli ligase.Even if only form flank by a base, also connect.When whole four kinds of dNTP exist, also connect, it does not limit the length of flank or the degree of gap translation, shows that the 5 ' phosphoric acid that is connected in 5 ' end of FAM oligonucleotide substrate produces and occurs immediately afterwards.

Example 5

Paired nick translation-ligation reaction using thermostable enzymes

Rationale: This experiment was performed to evaluate the effects of reaction temperature and the number of units of Taq DNA polymerase in paired reactions.

Material:

5' adapter oligonucleotide for gap translation (13-144) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Taq DNA ligase (New England BioLabs, catalog number M0208S)

10X Taq DNA ligase reaction buffer (New England BioLabs)

Taq DNA polymerase, concentrated 25 U/ul (Genscript, catalog number E00012)

method:

Reactions were assembled in a total volume of 30 μl containing a final concentration of 1× Taq DNA ligase reaction buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide for nick translation and 45 pmoles of oligonucleotide template, 200 μM of each of the following: dATP, dTTP, dGTP, or dTTP, 40 units of Taq DNA ligase, 80 units of Taq DNA ligase, or 120 units of Taq DNA ligase, and 10 units of Taq DNA polymerase. Reactions were incubated at 45°C, 50°C, 55°C, or 60°C for 30 minutes. 10 μl of those reactions were mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, visualized on a Dark Read light box (Clare Chemical Research) and photographed using a digital camera.

result:

Taq DNA polymerase extends the 3' hydroxyl end of 5' adapter oligonucleotide and removes the nucleotides on the FAM oligonucleotide substrate by its 5' flank endonuclease activity. Adding dTTP/dGTP/dATP (Figure 23, swimming lanes 2-5, Figure A) or dTTP (Figure 23, swimming lanes 6-9, Figure A) allows the addition of four and one bases at the 3' end of 5' adapter oligonucleotide, respectively, and the subsequent cutting of the 5' end of the FAM oligonucleotide substrate. At 60°C, the connection is unpaired (Figure 23, swimming lanes 5 and 9, Figure A). The connection efficiency is not affected by the addition of dTTP/dGTP/dATP (Figure 23, swimming lanes 2-5, Figure A) or dTTP (Figure 23, swimming lanes 6-9, Figure A). The connection efficiency depends on the amount of Taq DNA ligase present in the reaction. When 120 units of Taq DNA ligase were added to the reaction (Figure 23, lane 4, panel B), the abundance of ligation products was higher compared to 40 or 80 units (Figure 23, lanes 2 and 3, panel B, respectively). Lane 1, panel A, and lane 1, panel B show no enzyme control oligonucleotides.

in conclusion:

During the nick translation reaction, Taq DNA polymerase cleaves the 5' end of the FAM oligonucleotide substrate and generates the 5' phosphate termini required by Taq DNA ligase for ligation at 45°C to 60°C. This ligation is reduced at 60°C. The concentration of Taq DNA ligase in the reaction also affects ligation efficiency, as more product is observed in the presence of 120U of the enzyme compared to 80U and 40U.

Example 6

Paired displacement-cleavage-ligation reaction

Principle: This experiment was performed to show that thermostable Taq DNA ligase or thermolabile E. coli ligase can be combined with Taq DNA polymerase in paired displacement-cleavage ligation reactions.

Material:

5' adapter oligonucleotide for displacement-cleavage (13-156) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

Taq DNA ligase (New England BioLabs, catalog number M0208S)

10X Taq DNA ligase reaction buffer (New England BioLabs)

Taq DNA polymerase, concentrated 25 U/ul (Genscript, catalog number E00012)

Escherichia coli DNA ligase (New England BioLabs, catalog number M0205S)

10X E. coli DNA ligase reaction buffer (New England BioLabs)

method:

Reactions were assembled in a total volume of 30 μl containing a final concentration of 1× E. coli DNA ligase reaction buffer or 1× Taq DNA ligase reaction buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide for displacement-cleavage and 45 pmoles of oligonucleotide template, 10 units of E. coli DNA ligase or 40 units of Taq DNA ligase, and 10 units of Taq DNA polymerase. Reactions were incubated at 40°C or 45°C for 30 minutes. 10 μl of those reactions were mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, visualized on a Dark Read light box (Clare Chemical Research) and photographed using a digital camera.

result:

The 5' adapter oligonucleotide for displacement-cleavage has an extra matching base " T " at its 3' end, which overlaps with the 5' end of the FAM oligonucleotide substrate. When the 3' end of the 5' adapter oligonucleotide displaces the 5' end of the FAM oligonucleotide substrate, the 5' flanking endonuclease activity of Taq DNA polymerase cuts the 5' end of the FAM oligonucleotide substrate to produce a 5' phosphate, which is necessary for the connection using E. coli ligase (Figure 24, lane 2, Figure A) or Taq DNA ligase (Figure 24, lane 2, Figure B). Lane 1 of Figure A and B shows a no-enzyme oligonucleotide control.

in conclusion:

In the absence of dNTPs, no 5' adapter extension occurs. However, Taq DNA polymerase can cleave the 5' end of the FAM oligonucleotide substrate and generate a terminal 5' phosphate, which is required for ligation by E. coli DNA ligase or Taq DNA ligase.

Example 7

Paired displacement-cleavage-ligation reactions using "N" universal/degenerate or "T" substrate-specific 5' adapter 3' overhangs

Principle: This experiment shows that 5' adapter ligation using flanking endonucleases can be performed if the 5' adapter 3' end overhang is sequence-specifically matched or if it contains a degenerate non-sequence-specific 'N'.

Material:

5' adapter oligonucleotide for replacement-cleavage "T" (13-607) (Table 1)

5' adapter oligonucleotide for displacement-cleavage "N" (13-596) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

Taq DNA ligase (New England BioLabs, catalog number M0208S)

10X Taq DNA ligase reaction buffer (New England BioLabs)

Taq DNA polymerase, concentrated 25 U/ul (Genscript, catalog number E00012)

Escherichia coli DNA ligase (New England BioLabs, catalog number M0205S)

10X E. coli DNA ligase reaction buffer (New England BioLabs)

method:

Reactions were assembled in a total volume of 30 μl and contained a final concentration of 1× Taq DNA ligase reaction buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide "T" or 45 pmoles of 5′ adapter oligonucleotide "N" or 180 pmoles of 5′ adapter oligonucleotide "N" or 450 pmoles of 5′ adapter oligonucleotide "N" and 45 pmoles of oligonucleotide template, 40 units of Taq DNA ligase, and 10 units of Taq DNA polymerase. Reactions were incubated at 45°C, 50°C, or 55°C for 30 minutes or eight cycles of 45°C for 3 minutes and 65°C for 15 seconds. 10 μl of those reactions were mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven, visualized on a Dark Read light box (Clare Chemical Research) and photographed using a digital camera.

result:

When the 5' adapter oligonucleotide used for displacement-cleavage has a "T" that matches the oligonucleotide template at its 3' end (Figure 25, lanes 3, 5, 7, Figure A), (which overlaps with the 5' end of the FAM oligonucleotide substrate), ligation occurs at a higher rate than when the 5' adapter oligonucleotide has a degenerate "N" base, where all four nucleotides are present at that position during oligomer synthesis (Figure 25, lanes 2, 4, 6, Figure A), which is only a quarter of the time for a perfect match to the oligonucleotide template. Different reaction temperatures (45°C, 50°C, and 55°C) were tested, and no 5' adapter oligonucleotide "N" was used to improve ligation (Figure 25, lanes 2, 4, 6, Figure A). Similarly, different amounts of 5' adapter oligonucleotide "N" (45 pmoles, 180 pmoles, and 450 pmoles) were tested, without improving the ligation reaction (Figure 25, lanes 3-5, Figure B). However, temperature cycling of the reaction from 45°C to 65°C allowed ligation to occur at the highest rate, which was comparable to the "T"-matched 5' adapter oligonucleotide (Figure 25, lane 6, panel B). Lane 1 of panels A and B shows a no enzyme oligonucleotide control.

in conclusion:

To allow efficient 5' adapter ligation coupled with displacement-cleavage using the 5' adapter oligonucleotide "N," it is critical to cycle between a first temperature at which the Taq DNA ligase operates and a second temperature at which the duplex between the oligonucleotide template and the 5' adapter oligonucleotide "N" can separate. The cycling conditions allow for multiple associations between the 5' adapter oligonucleotide "N" and the oligonucleotide template, wherein the displacement-cleavage reaction only occurs if the 3' terminal base of the 5' adapter oligonucleotide is a perfect match to the template and can displace the 5' end of the FAM oligonucleotide substrate.

Example 8

Paired nick translation-ligation reaction using DNA polymerase I

Principle: This experiment shows that DNA polymerase I, which has 5'-3' exonuclease activity, can also participate in nick translation paired with the adapter ligation method.

Material:

5' adapter oligonucleotide for gap translation (13-144) (Table 1)

FAM oligonucleotide substrate (13-581) (Table 1)

Oligonucleotide template (13-582) (Table 1)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

25 bp ladder DNA size marker (Invitrogen (Life technologies), catalog number 10488-022)

Escherichia coli DNA ligase (Enzymatics, catalog number L6090L)

10X E. coli DNA ligase buffer (Enzymatics, catalog number B6090)

Taq-B DNA polymerase (Enzymatics, catalog number P7250L)

DNA polymerase I (New England BioLabs, catalog number M0209S)

method:

Reactions were assembled in a total volume of 30 μl containing a final concentration of 1× E. coli DNA ligase buffer, 30 pmoles of FAM oligonucleotide substrate, 45 pmoles of 5′ adapter oligonucleotide for nick translation and 45 pmoles of oligonucleotide template, 200 μM of each of the four dNTPs, 10 units of E. coli ligase, and either 10 units of Taq-B DNA polymerase or 5 units of DNA polymerase I or 1 unit of DNA polymerase I. Reactions were incubated at 40°C, 18°C, 16°C, or 14°C for 30 minutes. 10 μl of each reaction was mixed with 10 μl of 2x formamide loading buffer (97% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, and then run on a precast 15% polyacrylamide gel (TBE-Urea (Invitrogen, catalog number S11494)) in a 65°C oven and visualized with or without SYBR gold (upper and lower panels, respectively) in a Dark Read light box (Clare Chemical Research) and photographed with a digital camera.

result:

The first swimming lane of Figure 26 shows the enzyme-free control.In the presence of Taq-B archaeal dna polymerase and Escherichia coli ligase (Figure 26, swimming lane 2), 5 ' adapter oligonucleotide is connected to FAM oligonucleotide substrate, produces the product (Figure 26, swimming lane 2, upper figure and lower figure) of 69 bases, or extends completely, forms the new product (Figure 26, swimming lane 2, upper figure) of 58 bases.69 base products are from the extension carried out by Taq-B archaeal dna polymerase, and form flank at 5 ' end of FAM oligonucleotide substrate.Taq-B 5 ' flank endonuclease activity cuts flank and produces 5 ' phosphoric acid, which is utilized by Escherichia coli ligase, so that connection is complete.When FAM oligonucleotide substrate is completely replaced and degraded by 5 ' flank endonuclease activity of Taq-B archaeal dna polymerase in extension process, obtain the product of 58 bases. These two types of products are also formed when Taq-B DNA polymerase is replaced by DNA polymerase I ( FIG. 26 , lanes 3-8), which has 5′→3′ exonuclease activity that removes nucleotides from the head of the growing DNA chain one by one and allows nick translation to occur. The reaction was performed using 5 units of DNA polymerase I ( FIG. 26 , lanes 3-5) or 1 unit of DNA polymerase I ( FIG. 26 , lanes 6-8). Reactions using thermophilic Taq-B DNA polymerase were performed at 40° C. ( FIG. 26 , lane 2), while reactions using mesophilic DNA polymerase I were performed at 18° C. ( FIG. 26 , lanes 3 and 6), 16° C. ( FIG. 26 , lanes 4 and 7), or 14° C. ( FIG. 26 , lanes 5 and 8). In all cases, 69 bases of ligation products were obtained, but adding only 1 unit of DNA polymerase I (Figure 26, lanes 6-8) was more efficient than using 5 units (Figure 26, lanes 3-5). This explains the extremely strong 5'→3' exonuclease activity of DNA polymerase, which causes the rapid partial degradation of the FAM oligonucleotide substrate before it can be connected. Degradation products were observed at the bottom of the figure below (Figure 26, lanes 3-5). 25bp ladder DNA size marker was loaded in lane M.

in conclusion:

Taq-B DNA polymerase (thermophilic polymerase) and DNA polymerase I (normothermophilic polymerase) can both be used for the connection of gap translation mediation, but they require different conditions to fully bring into play activity.They all produce the product of 69 bases, which is the result of excising 5 ' end before connecting, but they adopt different mechanisms.Although Taq-B produces flanks, the flanks are cut to produce the end of required 5 ' phosphorylation for the connection by E. coli ligase, DNA polymerase I removes nucleotides one by one in front of the growing chain, and produces 5 ' phosphorylated nucleotides, which are the perfect substrates for E. coli ligase to engage two fragments.DNA polymerase I can be used for the 5 ' adapter body connection of gap translation mediation.

Example 9

Blunt-end ligation of physically sheared DNA requires polishing, and dephosphorylation prevents the formation of chimeric ligation products.

Principle: This experiment demonstrates the importance of end-polishing and dephosphorylation for blunt-end ligation of adapters to physically sheared DNA substrates.

Material:

Blue buffer (Enzymatics, catalog number B0110)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Adenosine 5′-triphosphate (ATP) (New England BioLabs, catalog number P0756S)

DNA polymerase I, large (Klenow) fragment (New England BioLabs, catalog number M0210S)

T4 DNA polymerase (New England BioLabs, catalog number M0203S)

T4 polynucleotide kinase (New England BioLabs, catalog number M0201S)

Shrimp alkaline phosphatase (Affymetrix, catalog number 78390)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

Escherichia coli ATCC 11303 genomic DNA (Affymetrix, catalog number 14380)

M220 Focused Ultrasound System, (Covaris, catalog number PN 500295)

Pippin Prep (Sage Science)

DNA Cleaner and Concentrator-5 (Zymo Research, catalog number D4004)

CDF2010 2% agarose, stain-free with internal standard (Sage Science)

method

E. coli gDNA was resuspended in DNA suspension buffer (Teknova, catalog number T0227) at a concentration of 100 ng/ul. DNA was fragmented to an average size of 150 base pairs using an M220 focused ultrasonicator. Subsequently, a Pippin Prep instrument was used to size-select the tightly distributed fragmented DNA from approximately 150 bp to approximately 185 bp on a 2% agarose gel.

In the reaction A group, 100ng or 500ng of size-selected DNA was subjected to the activity of the finishing enzyme. The reaction was assembled in a total volume of 30μl, which contained a final concentration of 1x Blue buffer, 100μM of each dNTP, 3 units of T4 DNA polymerase, 5 units of DNA polymerase I, large (Klenow) fragment, 1mM ATP, and 10 units of T4 polynucleotide kinase. The reaction was incubated at 30°C for 20 minutes. The DNA was purified using a DNA Clean & Concentrate-5 column. The DNA was eluted in 15μl of DNA suspension buffer and subjected to a subsequent dephosphorylation reaction B, followed by adapter ligation or direct insertion into the ligation reaction without dephosphorylation. The dephosphorylation reaction was assembled in a final volume of 30μl, including the treated DNA, 1x Blue buffer, and 1 unit of shrimp alkaline phosphatase. The reaction was incubated at 37°C for 10 minutes. The DNA was purified using a DNA Clean & Concentrate-5 column and eluted in 15μl of DNA suspension buffer.

In one set of reactions, 100 ng of size-selected DNA was dephosphorylated and then polished, or polished directly in one set of reactions, D. Dephosphorylation reactions were assembled in a final volume of 30 μl and included the processed DNA, 1× Blue buffer, and 1 unit of shrimp alkaline phosphatase. The reactions were incubated at 37°C for 10 minutes. DNA was purified using a DNA Clean & Concentrate-5 column and eluted in 15 μl of DNA suspension buffer. Polishing reactions, D, were assembled in a total volume of 30 μl and contained a final concentration of 1× Blue buffer, 100 μM of each dNTP, 3 units of T4 DNA polymerase, 5 units of DNA polymerase I, large (Klenow) fragment (lanes 6-7). DNA was purified using a DNA Clean & Concentrate-5 column and eluted in 15 μl of DNA suspension buffer.

The DNA of 50ng of each connection is ligated to 400ng of PBS.After purification, all previous reactions were ligated. The reaction was assembled in a final volume of 30 μ l, comprising the DNA, 1x T4 DNA ligase reaction buffer and 1200 units of T4 DNA ligase for processing. The reaction was incubated at 25°C for 15 minutes. The DNA from each 33ng connection was mixed with 2x formamide loading buffer (97% formamide, 10mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), heated at 95°C for 5 minutes, subsequently run on 15% polyacrylamide gels (TBE-Urea (Invitrogen, catalog number (Cat. No.) S11494)) prefabricated in 65°C stoves, stained with SYBR gold, observed and taken photos on a digital camera at Dark Reading light box (Clare Chemical Research Co., Ltd. (Clare Chemical Research)).

result:

Before finishing, physically sheared DNA is not a suitable substrate for the connection of blunt-ended adapters by T4 DNA ligase (Figure 27, lane 1). After finishing with T4 polynucleotide kinase, T4 DNA polymerase and Klenow fragment, the DNA ends are blunt, some 5' ends are phosphorylated, and the molecules can link or connect to each other and link or connect to blunt adapters (Figure 27, lanes 2 and 4). Materials of about 325 bases, about 500 bases and more than 500 bases correspond to the connection of 2 molecules, 3 molecules and 4 molecules of about 175 bases together, respectively (Figure 27, lanes 2 and 4). The concentration of DNA affects the formation of the connection product. When higher concentrations of DNA are present, the abundance of higher molecular weight chimeric connection materials is higher (Figure 27, lane 4). After the finishing step, DNA treatment with shrimp alkaline phosphatase affects the formation of concatemers between DNA molecules (Figure 27, lanes 3 and 5). Treatment with shrimp alkaline phosphatase also prevented concatemer formation if performed before polishing of the fragmented DNA ( FIG. 27 , lane 6). The abundance of ligation products observed after polishing with T4 DNA polymerase and Klenow fragment ( FIG. 27 , lane 7) was lower than that observed after polishing with T4 DNA polymerase, Klenow, and T4 polynucleotide kinase ( FIG. 27 , lane 2).

in conclusion:

The blunt connection efficiency of the DNA of physical shearing depends on the end finishing carried out by DNA polymerase.The connection is also improved by adding T4 polynucleotide kinase, which phosphorylates the 5' ends of the DNA fragments and dephosphorylates the 3' ends.The concentration of DNA also affects the amount of connection and the formation of chimeric products.At higher concentrations, DNA is more likely to form chimeric products in the presence of T4 DNA ligase.Alkaline phosphatase removes 5' phosphate (which is required for connection) and prevents the formation of chimeric connection products (concatemers).

Example 10

NGS libraries have increased yield when prepared using 5’ base trimming paired with adapter ligation reactions.

Principle: This experiment demonstrates its exemplary application to the reactions present in NGS library construction, specifically the increase in library yield obtained by including 5' base trimming paired with 5' adapter ligation. The library is constructed from size-selected sheared DNA, so the library products can be easily visualized by gel electrophoresis.

Material:

Blue buffer (Enzymatics, catalog number B0110)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Adenosine 5′-triphosphate (ATP) (New England BioLabs, catalog number P0756S)

Klenow fragment (Enzymatics, catalog number P7060L)

T4 DNA polymerase (Enzymatics, catalog number P7080L)

T4 polynucleotide kinase (Enzymatics, catalog number Y904L)

Shrimp alkaline phosphatase (Affymetrix, catalog number 78390)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

3' adapter; first oligonucleotide 13-501 (Table 1)

3' adapter; second oligonucleotide 13-712 (Table 1)

Escherichia coli ATCC 11303 genomic DNA (Affymetrix, catalog number 14380)

M220 focused ultrasound system (Covaris, catalog number PN 500295)

Escherichia coli DNA ligase (Enzymatics, catalog number L6090L)

E. coli DNA ligase buffer (Enzymatics, catalog number B6090)

Uracil-DNA glycosidase (Enzymatics, catalog number G5010L)

Taq-B DNA polymerase (Enzymatics, catalog number P7250L)

5' adapter oligonucleotide for gap translation (13-489) (Table 1)

5' adapter oligonucleotide for displacement-cleavage (13-595) (Table 1)

Taq DNA ligase (Enzymatics, catalog number L6060L)

SPRIselect (Beckman Coulter, catalog number B23419)

method:

Escherichia coli genomic DNA was resuspended in DNA suspension buffer (Teknova, catalog number T0227) at a concentration of 100 ng/μl. DNA was fragmented into an average size of 150 base pairs using an M220 focused ultrasound instrument. Subsequently, a Pippin Prep instrument was used to size-select the tightly distributed fragmented DNA from approximately 150 bp to approximately 185 bp on a 2% agarose gel.

The library was prepared using an enhanced adapter ligation method using 100 ng of size-selected E. coli genomic DNA. The polishing reaction was assembled in 30 μl and contained a final concentration of 1x Blue buffer, 100 μM of each dNTP, 3 units of T4 DNA polymerase, 5 units of DNA polymerase I, large (Klenow) fragment, and 10 units of T4 polynucleotide kinase. The reaction was incubated at 37°C for 20 minutes. The DNA was purified using DNA Clean & Concentrate-5 and eluted in 15 μl of DNA Suspension Buffer. The 3' adapter ligation reaction was assembled in 30 μl and included 1x T4 DNA ligase buffer, 220 pmoles of 3' adapter first oligonucleotide, 440 pmoles of 3' adapter second oligonucleotide, 15 μl of purified DNA, and 1200 units of T4 DNA ligase. The reaction was incubated at 25°C for 15 minutes. DNA was placed in a volume of up to 50 μl and purified and size-selected using 70 μl SPRIselect beads (ratio 1.4x). DNA was eluted in 15 μl of DNA resuspension buffer. Partial degradation of the 3' adapter, annealing of the 5' adapter, 5' end trimming and ligation of the 5' adapter all occurred in the next reaction, which was assembled in a final volume of 30 μl and contained 1x E. coli DNA ligase buffer or 1x Taq DNA ligase buffer, 200 μM of each dNTP or 200 μM of each dATP, dTTP, dGTP or no dNTP, 200 pmoles of 5' adapter oligonucleotide for gap translation or 5' adapter oligonucleotide for displacement-cleavage, 10 units of E. coli ligase or 40 units of Taq DNA ligase, 2 units of uracil-DNA glycosidase, 10 units of Taq-B DNA polymerase and 15 μl of DNA purified after the 3' adapter ligation reaction. Reactions were incubated at 40°C or 45°C for 10 minutes or 30 cycles of 45°C for 45 seconds to 65°C for 5 seconds (library 5). DNA was placed in a volume of up to 50 μl and purified and size-selected using 40 μl of SPRIselect beads (ratio 0.8x). DNA was eluted in 20 μl and quantified by qPCR using the Kapa Library Quantification Kit - Illumina/Universal (Cat. No. KK4824).

result:

The library concentrations are reported in the figure (Figure 28, Panel A) and the libraries were visualized by electrophoresis under denaturing conditions on a 6% polyacrylamide gel (Figure 28, Panel B). The input DNA migrated between about 150 bases and about 185 bases (Figure 28, lane I, Panel B). An aliquot was taken after the 3' adapter ligation step and loaded on the gel. The product migrated between about 225 and about 250 bases, which corresponds to the addition of 64 bases of the 3' adapter (Figure 28, lane L, Panel B). The contribution of Taq-B DNA polymerase in removing one or more bases and exposing the 5' phosphate group at the 5' end of the DNA before ligation of the 5' adapter is shown in Library 1 vs. 2 (Figure 28, lanes 1 and 2, Panels A and B). The concentration of Library 1 made without Taq-B (2.6 nM) was one-third that of Library 2 made with Taq-B DNA polymerase (7.9 nM). Even after treatment with T4 polynucleotide kinase, 75% of the fragmented DNA required processing of its 5' end to become compatible for connection. The completed libraries were also loaded on the gel (Figure 28, lanes 1 and 2, Figure B). These libraries migrated between about 275 bases and about 300 bases, corresponding to the addition of 58 bases for gap translation of the 5' adapter oligonucleotide or the 5' adapter oligonucleotide for displacement-cutting and the 3' adapter of 64 bases. The intensity of the library 1 product display was lower than that of the band of library 2 (Figure 28, Figure B). During the partial degradation of the 3' adapter, the annealing of the 5' adapter, the 5' end trimming and the connection process of the 5' adapter step, libraries 3 and 4 were prepared using dATP, dTTP, dGTP and E. coli ligase or Taq DNA ligase, respectively. The concentration of library 3 (4.8nM) was about 60% of that of library 2 (7.9nM). This 30% loss in yield is related to the percentage of thymine "C" in the E. coli genome (25%). Each time the 5' end of the DNA substrate is a thymine, the 5' adapter oligonucleotide used for gap translation cannot be extended by Taq and the 5' end cannot be trimmed. There are also additional 6.25% and 1.5% possibilities to have two and three consecutive thymines at the 5' end of the DNA substrate, respectively. When compared with E. coli ligase at 40°C (5.2nM) (library 3), ligation using Taq DNA ligase at 45°C (library 4) obtained a similar yield (4.8nM). Library 5, which was prepared using a 5' adapter oligonucleotide for displacement-cleavage, (4.2nM) was less efficient than library 2 (7.9nM) prepared using a 5' adapter oligonucleotide for gap translation.

in conclusion:

The library was successfully prepared using the adapter ligation method disclosed herein. Compared to libraries without a 5' end processing step, 5' end DNA trimming by Taq DNA polymerase allowed a threefold increase in the yield of 5' adapter ligation products (library 1 vs. 2). Both Taq DNA ligase (library 4) and E. coli ligase (library 3) efficiently connected 5' adapters after gap translation. Taq DNA ligase specifically connected 5' adapters after displacement-cleavage (library 5). Using 4 dNTPs (library 2) instead of 3 (libraries 3 and 4) during gap translation allows for the connection of more DNA substrates to the 5' adapter.

Example 11

Sequence analysis of NGS libraries prepared by 5’ base trimming paired with adapter ligation

Principle: This experiment demonstrates the application of the present reaction in its exemplary application to NGS library construction. Libraries were constructed from sheared E. coli DNA and then sequenced to demonstrate the excellent uniformity of coverage obtained over a wide base composition range of the genome.

Material:

Blue buffer (Enzymatics, catalog number B0110)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

100 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) sets, PCR grade (Invitrogen (Life technologies), catalog number 10297-018)

Adenosine 5′-triphosphate (ATP) (New England BioLabs, catalog number P0756S)

Klenow fragment (Enzymatics, catalog number P7060L)

T4 DNA polymerase (Enzymatics, catalog number P7080L)

T4 polynucleotide kinase (Enzymatics, catalog number Y904L)

Shrimp alkaline phosphatase (Affymetrix, catalog number 78390)

T4 DNA ligase (Fast) (Enzymatics, catalog number L6030-HC-L)

10X T4 DNA ligase buffer (Enzymatics, catalog number B6030)

3' adapter; first oligonucleotide 13-510 (Table 1)

3' adapter; second oligonucleotide 13-712 (Table 1)

Escherichia coli ATCC 11303 genomic DNA (Affymetrix, catalog number 14380)

M220 focused ultrasound system (Covaris, catalog number PN 500295)

Escherichia coli DNA ligase (Enzymatics, catalog number L6090L)

E. coli DNA ligase buffer (Enzymatics, catalog number B6090)

Uracil-DNA glycosidase (Enzymatics, catalog number G5010L)

Taq-B DNA polymerase (Enzymatics, catalog number P7250L)

5' adapter oligonucleotide for gap translation (13-489)

SPRIselect (Beckman Coulter, catalog number B23419)

method:

Escherichia coli genomic DNA was resuspended in DNA suspension buffer (Teknova, catalog number T0227) at a concentration of 100 ng/μl. DNA was fragmented into an average size of 150 base pairs using an M220 focused sonicator. 100 ng of Escherichia coli Covaris genomic DNA was used to prepare the library. The first dephosphorylation reaction was assembled in a total volume of 15 μl and contained a final concentration of 1x Blue buffer, 100 ng of fragmented Escherichia coli genomic DNA, and 1 unit of shrimp alkaline phosphatase. The reaction was incubated at 37°C for 10 minutes. Shrimp alkaline phosphatase was inactivated at 65°C for 5 minutes. The polishing reaction was assembled in 30 μl and contained a final concentration of 1x Blue buffer, 100 μM of each dNTP, 3 units of T4 DNA polymerase, 5 units of DNA polymerase I, large (Klenow) fragment, and 15 μl of dephosphorylation reaction. The reaction was incubated at 20°C for 30 minutes. DNA was purified using DNA Clean & Concentrator-5. DNA was eluted in 15 μl of DNA Resuspension Buffer. 3' adapter ligation reactions were assembled in 30 μl of 1x T4 DNA Ligase Buffer, 220 pmoles of 3' adapter oligonucleotide 1, 440 pmoles of 3' adapter oligonucleotide 2, 15 μl of polished purified DNA, and 1200 units of T4 DNA Ligase. The reactions were incubated at 25°C for 15 minutes. After adjusting the volume to 50 μl, DNA was purified and size-selected using 45 μl of SPRIselect beads (ratio 0.9x). DNA was eluted in 15 μl of DNA Resuspension Buffer. Partial degradation of the 3' adapter, annealing of the 5' adapter, trimming of the 5' end DNA of the 5' adapter and ligation all occurred in the next reaction, which was assembled in a final volume of 30 μl and contained 1x E. coli DNA ligase, 200 μM of each dNTP, 200 pmoles of 5' adapter oligonucleotide for gap translation, 10 units of E. coli ligase, 2 units of uracil-DNA glycosidase, 10 units of Taq-B DNA polymerase and 15 μl of DNA purified after the 3' adapter ligation reaction. The reaction was incubated at 40°C for 10 minutes. After adjusting the volume to 50 μl, the DNA was purified using 70 μl of SPRIselect beads (ratio 1.4x). The DNA was eluted in 20 μl and quantified by qPCR using the Kapa Library Quantification Kit-Illumina/Universal (Cat. No. KK4824). DNA was denatured with sodium hydroxide at a final concentration of 0.1 mM for 5 minutes, and 600 μl of 10 pM library was loaded on MiSeq (Illumina).

result:

The library concentration was quantified as 2.8 nM by qPCR. The double-end reads of 76 bases were produced by the v2 chemical method of Illumina MiSeq. 928K/mm ² clusters were produced, and the Q30 scores of the first and second readings were 97.8% and 96.9% respectively. The sequence data quality was assessed using FastQC reports (Babraham Bioinformatics). The summary of the analysis showed 9 green tick marks, 2 yellow exclamation marks (warnings), but no red X (failure) was observed (Figure 29, Figure A). The overall %GC of all bases in all sequences was 50%, as expected for the E. coli genome (green tick mark, Figure 29, Figure B). The quality of the sequence of each reading of the 76 bases analyzed was excellent (green tick mark, Figure 29, Figure C). The percentage of each base is plotted in Figure D. At any reading, the amount of G/C and A/T has a difference of <10% (green tick mark, Figure 29, Figure D). The GC content of the 76 bases analyzed is similar (green tick mark, Figure 29, Figure E). The GC content of each reading throughout the length of each sequence is compared with the theoretical distribution (yellow exclamation mark, Figure 29, Figure F). The reminder appears because the sum of the differences from the normal distribution is found in more than 15% of the readings (yellow exclamation mark, Figure 29, Figure F). No reminder appears for the N content per base or sequence length distribution (summary, Figure 29, Figure A). The sequence duplication level is 35.85% (Figure 29, Figure G). The yellow warning appears because non-unique sequences make up more than 20% of the total amount, which is attributed to the high level of coverage 135x (yellow exclamation mark, Figure 29, Figure G). No sequences or kmers that appear too much are reported (summary, Figure 29, Figure A). In fact, no adapter dimers are observed (0.02%, data not shown). GC bias was assessed using Picard CollectGcBiasMetrics. Coverage uniformity was maintained across a wide range of base compositions. Coverage bias was only observed below 10% GC content or above 80%. Base quality exceeded Q25, which corresponds to 99.8% accuracy in base calling. Similarly, lower quality was only observed at very low and high GC contents.

in conclusion:

Libraries were successfully prepared using fragmented E. coli genomic DNA. Sequencing revealed high-quality data with unbiased coverage across the entire GC content range.

Example 12

Comprehensive oncology heat map with TP53 gene coverage

Principle: A total of 51 amplicons were designed to cover the entire coding region of the TP53 gene as well as 30 hotspot loci representing clinically actionable mutations in oncology.

Principle: This amplicon plot provides proof of concept for the method described herein, with significant overlap among the 51 amplicons demonstrating the lack of small amplicons dominating the reaction and the uniformity of coverage across amplicons achievable with limited multiplex cycle numbers. Furthermore, the high percentage of on-target reads demonstrates the specificity of primer action, as primer-dimers and nonspecific off-target amplification products are absent from the sequencing library.

Material:

*Human HapMap genomic DNA (Coriell Institute, NA12878)

*KAPA High Fidelity Hot Start Uracil + ReadyMix (KAPA Biosystems, catalog number KK2802)

*102 target-specific primers (Table 2)

*Universal primer containing the 3' adapter oligonucleotide truncated sequence and cleavable bases 14-882 (Table 2)

*E. coli DNA ligase buffer (Enzymatics, catalog number B6090)

*5’ adapter oligonucleotide, used for adapter ligation step (14-571)

*5' portion of the 3' adapter oligonucleotide, used in the adapter ligation step (14-877)

*Adapter oligonucleotides for adapter ligation steps 14-382 (Table 2)

*E. coli DNA ligase (Enzymatics, catalog number L6090L)

*Uracil-DNA glycosidase (Enzymatics, catalog number G5010L)

*Endonuclease VIII (Enzymatics, catalog number Y9080L)

*Taq-B DNA polymerase (Enzymatics, catalog number P7250L)

*SPRIselect (Beckman Coulter, catalog number B23419)

*20% PEG-8000/2.5M NaCl solution for purification steps

method:

Human genomic DNA was diluted in DNA suspension buffer (Teknova, catalog number T0227) at a concentration of 2 ng/μl. DNA was gently sheared by vortexing for 2 minutes. 10 ng of the sheared genomic DNA was used to prepare the library. The first reaction of amplification was assembled in a total volume of 30 μl and contained a mixture of 102 target-specific primers in different proportions at a final concentration of 1x KAPA high-fidelity hot start uracil+ReadyMix, 10 ng of sheared human genomic DNA, 300 pmol of universal primers, and a final concentration of 0.85 uM. The reaction was run through a cycling program of 95°C for 3 minutes, followed by 4 cycles of 98°C for 20 seconds, 63°C for 5 minutes, and 72°C for 1 minute to produce target-specific amplicons, and terminated by 23 cycles of 98°C for 20 seconds and 64°C for 1 minute to produce multiple copies of target-specific amplicons. After adjusting the volume to 50 μl, the DNA product was purified using 60 μl of SPRIselect beads (ratio 1.2x). The beads were resuspended in 50 μl of 1x reaction mixture, which contained 1x E. coli ligase buffer, 100 pmol of adapter oligonucleotides, 10 units of E. coli ligase, 10 units of endonuclease VIII, 2 units of uracil-DNA glycosidase, 20 units of Taq-B DNA polymerase, 100 pmol of 5' adapter oligonucleotides and 100 pmol of 3' adapter oligonucleotides. The reaction was incubated at 37°C for 10 minutes and then purified by adding 42.5 μl of 20% PEG-8000/2.5M NaCl solution (ratio 0.85x). DNA was eluted in 20 μl and quantified by qPCR using Kapa Library Quantification Kit-Illumina/Universal (Cat. No. KK4824). DNA was denatured with sodium hydroxide at a final concentration of 0.1 mM for 5 minutes, and 600 μl of 10 pM library was loaded on MiSeq (Illumina).

result:

The library concentration was quantified as 19.1 nM by qPCR. Paired-end reads of 101 bases were generated using the v2 chemistry of the Illumina MiSeq. Prior to data analysis, sequence-specific trimming was performed from the 5' ends of reads 1 and 2 to remove synthetic primer sequences using the Cutadapt program. Paired read alignments to the human genome and to the target region using the BWA-MEM tool showed exceptional quality data, with 98% aligned to the target region. Coverage data was also obtained using the BED tool. Coverage uniformity was 100%, indicating that each of the 51 amplicons was present in the final library. The coverage of each individual base in each amplicon was also calculated and was higher than the average per-base coverage of 20%, indicating that none of the 51 amplicons was underrepresented in the final product. Figure 45 describes the coverage obtained for overlapping amplicons covering the coding exons of the TP53 gene. Figure 46 describes variant identification at an 18% frequency obtained by sequence analysis using VarScan and SAMtools.

in conclusion:

A targeted amplicon library was successfully prepared using human genomic DNA, and sequencing revealed high-quality data.

Table 1.

*:phosphorothioated DNA bases

/5SpC3/: 5’C3 spacer (IDT)

/3SpC3/: 3’C3 spacer (IDT)

/5PHOS/: 5' phosphorylation (IDT)

/3PHOS/: 3' phosphorylation (IDT)

/5FAM/:5’6-carboxyfluorescein (IDT)

/3FAM/:3’6-carboxyfluorescein (IDT)

ddT:2',3'-dideoxythymidine (TriLink)

Table 2 Oligonucleotides used in Example 12

*:phosphorothioated DNA bases (IDT)

/3PHOS/: 3' phosphorylation (IDT)

The foregoing disclosure is supplemented by the following description of various aspects and embodiments of the present invention, which are provided in the following enumerated paragraphs.

1. A method for producing a processed substrate molecule, the method comprising:

(i) ligating a first polynucleotide to the 3' end of a substrate molecule, said substrate molecule being at least partially double-stranded;

(ii) annealing the second polynucleotide to the first polynucleotide under conditions that promote annealing;

(iii) cleaving at least one nucleotide from the 5' end of the substrate molecule; and then

(iv) ligating the second polynucleotide to the 5' end of the double-stranded substrate molecule to produce the processed substrate molecule.

2. The method according to paragraph 1, further comprising, before step (i), contacting the substrate molecule with a phosphatase.

3. The method of paragraph 2, further comprising the step of making the substrate molecule have a blunt end by contacting the substrate molecule with a polymerase having 3'-5' exonuclease activity.

4. The method of paragraph 3, further comprising the step of contacting the substrate molecule with a template-independent polymerase to adenylate the 3' end of the substrate molecule.

5. The method of any one of paragraphs 1-4, wherein the substrate molecule is naturally occurring, or the substrate molecule is synthetic.

6. The method of paragraph 5, wherein the substrate molecule is naturally occurring.

7. The method of paragraph 6, wherein the substrate molecule is genomic DNA.

8. The method of paragraph 7, wherein the genomic DNA is eukaryotic or prokaryotic.

9. The method of paragraph 7 or paragraph 8, wherein the genomic DNA is fragmented in vivo or in vitro.

10. The method of paragraph 9, wherein the in vitro fragmentation is performed by a method selected from the group consisting of shearing, cleavage with an endonuclease, sonication, heating, irradiation with an α, β or γ source, chemical cleavage in the presence of metal ions, free radical cleavage, and combinations thereof.

11. The method of paragraph 9, wherein the in vivo fragmentation occurs by a method selected from the group consisting of apoptosis, irradiation, and asbestos exposure.

12. The method of paragraph 5, wherein the substrate molecule is synthetic and selected from the group consisting of cDNA, DNA produced by whole genome amplification, primer extension products comprising at least one double-stranded end, and PCR amplicons.

13. The method of any of paragraphs 1-12, wherein the first polynucleotide is at least partially double-stranded and comprises oligonucleotide 1 and oligonucleotide 2.

14. The method of paragraph 13, wherein the second polynucleotide anneals to oligonucleotide 1.

15. The method of paragraph 14, wherein the annealing results in a gap, a gap, or overlapping bases between the second polynucleotide and the substrate molecule.

16. The method of paragraph 14 or paragraph 15, wherein contacting the second polynucleotide with a polymerase results in degradation of Oligonucleotide 2.

15. The method of any of paragraphs 13-16, wherein oligonucleotide 2 comprises a base that is susceptible to degradation.

16. The method of any of paragraphs 13-17, wherein oligonucleotide 2 comprises a blocking group at its 3' end that prevents ligation.

17. The method of any of paragraphs 1-16, wherein the second polynucleotide comprises modified bases.

18. The method of paragraph 14, wherein the annealing results in dehybridization of Oligonucleotide 1 and Oligonucleotide 2.

19. The method of any one of paragraphs 1 to 18, further comprising:

(i) ligating a third polynucleotide to the 3' end of another at least partially double-stranded substrate molecule;

(ii) annealing the fourth polynucleotide to the third polynucleotide under conditions that promote annealing;

(iii) cleaving at least one nucleotide from the 5' end of the other substrate molecule; and then

(iv) ligating the fourth polynucleotide to the 5' end of another substrate molecule of the double strand to generate another processed substrate molecule.

20. The method of paragraph 19, wherein the first polynucleotide and the third polynucleotide are identical.

21. The method of paragraph 19 or paragraph 20, wherein the second polynucleotide and the fourth polynucleotide are identical.

Claims (40)

1. A next-generation sequencing adaptor system, comprising: A 3' linker polynucleotide comprising a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises a second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5' phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3' end, wherein the blocking group is not capable of linking. Phosphatases are used to remove phosphate from the 5' end of substrate nucleic acid molecules; DNA polymerases with 3'-5' exonuclease activity are used to fill the 3' recessed end of the substrate nucleic acid molecule and cleave the 3' protrusion. At least one 2'-deoxynucleoside 5'-triphosphate (dNTP); The first ligase used to link 3' adaptor polynucleotides to the 3' end of substrate nucleic acid molecules; 5' Integral polynucleotide, comprising a segment portion or a first sequence that is completely complementary to the first oligonucleotide of the 3' integral polynucleotide; A second ligase used to link 5' adaptor polynucleotides to the 5' end of substrate nucleic acid molecules; and DNA polymerase with nick translation activity. 2. The linker system of claim 1, wherein the second oligonucleotide comprises a readily degradable base, and wherein the readily degradable base is selected from deoxyuracil, ribonucleotide, deoxyhypoxanthin, and inosine. 3. The adaptor system of claim 2 further comprises an endonuclease that recognizes easily degradable bases. 4. The adapter system of claim 3, wherein the endonuclease is selected from the group consisting of: UDG plus endonuclease VIII, RNase HI, RNase H2 and endonuclease V. 5. The linker system of claim 1, wherein the first ligase is selected from Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase and T7 ligase, and wherein the second ligase is selected from Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase, T7 ligase, Taq ligase and Amp ligase. 6. The linker system of claim 1, wherein the blocking group is selected from 3'-deoxythymidine, 3'-deoxyadenine, 3'-deoxyguanine, 3'-deoxycytosine and 2'3'-dideoxynucleotide. 7. The linker system of claim 1, wherein the 5' linker polynucleotide is single-stranded. 8. The linker system of claim 1, wherein the second ligase is the same as the first ligase. 9. A next-generation sequencing adaptor system, comprising: A 3' linker polynucleotide comprising a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises a second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5' phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3' end, wherein the blocking group is not capable of linking. 5' Integral polynucleotide, comprising a segment portion or a first sequence that is completely complementary to the first oligonucleotide of the 3' integral polynucleotide; The first ligase used to link 3' adaptor polynucleotides to the 3' end of substrate nucleic acid molecules; A second ligase used to link 5' adaptor polynucleotides to the 5' end of substrate nucleic acid molecules; At least one 2'-deoxynucleoside 5'-triphosphate (dNTP); and DNA polymerase with nick translation activity. 10. A method for generating processed substrate molecules, the method comprising: (i) Applying a phosphatase to a sample containing a substrate nucleic acid molecule, wherein the substrate nucleic acid molecule is double-stranded; (ii) Incubate the sample under conditions that allow phosphatases to remove phosphate from the 5' end of the substrate nucleic acid molecule; (iii) Apply DNA polymerase and dNTPs with 3'-5' exonuclease activity to the sample; (iv) Incubate the sample under conditions that allow DNA polymerase with 3'-5' exonuclease activity to generate blunt ends on the substrate nucleic acid molecule; (v) Applying a 3' inverse polynucleotide and a first ligase to a sample, wherein the 3' inverse polynucleotide comprises a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises a second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5' phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3' end, wherein the blocking group is incapable of ligation; and (vi) Incubate the sample under conditions that sufficiently allow the first oligonucleotide to be attached to the 3' end of the substrate molecule to produce a first processed substrate nucleic acid molecule; (vii) Apply a 5' inverse polynucleotide, a second ligase, at least one dNTP, and a DNA polymerase with nick translation activity to a sample, wherein the 5' inverse polynucleotide comprises a first sequence that is either a segment portion of or completely complementary to the first oligonucleotide of the 3' inverse polynucleotide; and (viii) Incubate the sample under conditions sufficient to (a) promote the annealing of the 5' adaptor polynucleotide to the 3' adaptor polynucleotide; (b) allow the nick translation activity of the DNA polymerase; and (c) allow the 5' adaptor polynucleotide to be attached to the 5' end of the first processed substrate nucleic acid molecule, wherein after step (iv) is completed, the first processed substrate nucleic acid molecule is a second processed substrate nucleic acid molecule containing a 3' adaptor polynucleotide and a 5' adaptor polynucleotide attached to the 3' end and 5' end of the two strands of the substrate nucleic acid molecule, respectively. 11. A method for generating processed substrate molecules, the method comprising: (i) Applying a 3' adaptor polynucleotide and a first ligase to a sample containing a substrate nucleic acid molecule, wherein the substrate nucleic acid molecule is double-stranded, wherein the 3' adaptor polynucleotide comprises a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises a second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5' phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3' end, wherein the blocking group is not capable of ligation; (ii) Incubate the sample under conditions that sufficiently allow the first oligonucleotide to be attached to the 3' end of the substrate molecule to produce a first processed substrate nucleic acid molecule; (iii) Applying a 5' inverse polynucleotide, a second ligase, at least one dNTP, and a DNA polymerase with nick translation activity to a sample, wherein the 5' inverse polynucleotide comprises a first sequence that is either a segment portion of or completely complementary to the first oligonucleotide of the 3' inverse polynucleotide; and (iv) Incubate the sample under conditions sufficient to (a) promote the annealing of the 5' adaptor polynucleotide to the 3' adaptor polynucleotide; (b) allow the nick translation activity of the DNA polymerase; and (c) allow the 5' adaptor polynucleotide to be attached to the 5' end of the first processed substrate nucleic acid molecule, wherein after step (iv) is completed, the first processed substrate nucleic acid molecule is a second processed substrate nucleic acid molecule containing a 3' adaptor polynucleotide and a 5' adaptor polynucleotide attached to the 3' end and 5' end of the two strands of the substrate nucleic acid molecule, respectively. 12. The method of claim 10, wherein the phosphatase is bovine intestinal alkaline phosphatase or shrimp alkaline phosphatase. 13. The method of claim 10, further comprising: (iv-a) Apply a template-independent polymerase to the sample to adenylate the 3' end of the substrate nucleic acid molecule, wherein step (iv-a) is performed after step (iv) and before step (v). 14. The method of claim 10, further comprising, prior to step (i), fragmenting the substrate nucleic acid molecule by a process selected from the group consisting of: shearing, cutting with a nuclease, acoustic treatment, heating, chemical cutting, and combinations thereof, wherein the substrate nucleic acid molecule is genomic DNA. 15. The method of claim 10, wherein the substrate nucleic acid molecule is genomic DNA or wherein the substrate nucleic acid molecule is synthetic and selected from the group consisting of: cDNA, DNA generated by whole-genome amplification, primer extension products containing at least one double-stranded end, and PCR amplicon. 16. The method of claim 10, wherein the substrate nucleic acid molecule is genomic DNA or wherein the substrate nucleic acid molecule is synthetic and selected from the group consisting of: cDNA, DNA generated by whole-genome amplification, primer extension products containing at least one double-stranded end, and PCR amplicon. 17. The method of claim 10, wherein the substrate nucleic acid molecule is circulating cell-free DNA. 18. The method of claim 10, wherein the first ligase is selected from Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase and T7 ligase. 19. The method of claim 11, wherein the substrate nucleic acid molecule is circulating cell-free DNA. 20. The method of claim 11, wherein the first ligase is selected from Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase and T7 ligase. 21. The method of claim 10, wherein the second oligonucleotide comprises a readily degradable base, and wherein the readily degradable base is selected from deoxyuridine, ribonucleotide, deoxyhypoxanthin, and inosine. 22. The method of claim 21, further comprising the following step after step (vi): (vi-a) Applying a nuclease to the sample, wherein the nuclease recognizes the readily degradable bases; and (vi-b) Incubate the sample under conditions that allow endonucleases sufficient to degrade the readily degradable bases. 23. The method of claim 22, wherein the endonuclease is selected from the group consisting of: UDG+ endonuclease VIII, RNase HI, RNase H2, and endonuclease V. 24. The method of claim 10, wherein the second ligase is selected from the group consisting of Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase, T7 ligase, Amp ligase, and Taq ligase. 25. The method of claim 10, wherein the 5' connector is single-chained. 26. The method of claim 10, further comprising purifying the substrate nucleic acid molecule after one or more of steps (ii), (iv), (vi), and (viii). 27. The method of claim 10, wherein the condition for sufficient step (viii) includes incubation at 40°C for at least 10 minutes. 28. The method of claim 10, wherein the conditions for sufficient step (ii) include incubation at 37°C for at least 10 minutes. 29. The method of claim 10, wherein the condition for sufficient step (iv) includes incubation at 20°C for at least 10 minutes. 30. The method of claim 10, wherein the conditions for sufficient step (vi) include incubation at 25°C for at least 15 minutes. 31. The method of claim 10, wherein the blocking group is selected from 3'-deoxythymidine, 3'-deoxyadenine, 3'-deoxyguanine, 3'-deoxycytosine and 2'3'-dideoxynucleotide. 32. The method of claim 11, wherein the second oligonucleotide comprises a readily degradable base, and wherein the readily degradable base is selected from deoxyuracil, ribonucleotide, deoxyhypoxanthin, and inosine. 33. The method of claim 32, further comprising the following step after step (ii): (ii-a) Applying a nuclease to the sample, wherein the nuclease recognizes the readily degradable bases; and (ii-b) Incubate the sample under conditions that allow endonucleases sufficient to degrade the readily degradable bases. 34. The method of claim 33, wherein the endonuclease is selected from the group consisting of: UDG+ endonuclease VIII, RNase HI, RNase H2, and endonuclease V. 35. The method of claim 11, wherein the second ligase is selected from the group consisting of Escherichia coli DNA ligase, T4 DNA ligase, T3 ligase, T7 ligase, Amp ligase, and Taq ligase. 36. The method of claim 11, wherein the 5' connector is single-chained. 37. The method of claim 11, further comprising purifying the substrate nucleic acid molecule after one or more of steps (ii) and (iv). 38. The method of claim 11, wherein the condition for sufficient step (iv) includes incubation at 40°C for at least 10 minutes. 39. The method of claim 11, wherein the condition for sufficient step (ii) includes incubation at 25°C for at least 10 minutes. 40. The method of claim 11, wherein the blocking group is selected from 3'-deoxythymidine, 3'-deoxyadenine, 3'-deoxyguanine, 3'-deoxycytosine and 2'3'-dideoxynucleotide.