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WO2012116661A1 - Étiquette à adn et son utilisation - Google Patents

Étiquette à adn et son utilisation Download PDF

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Publication number
WO2012116661A1
WO2012116661A1 PCT/CN2012/071893 CN2012071893W WO2012116661A1 WO 2012116661 A1 WO2012116661 A1 WO 2012116661A1 CN 2012071893 W CN2012071893 W CN 2012071893W WO 2012116661 A1 WO2012116661 A1 WO 2012116661A1
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Prior art keywords
dna
tag
sequencing
present
library
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PCT/CN2012/071893
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English (en)
Chinese (zh)
Inventor
刘琳
何毅敏
杨焕明
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Publication of WO2012116661A1 publication Critical patent/WO2012116661A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags

Definitions

  • the invention relates to the field of nucleic acid sequencing technology, in particular to the field of multiplex nucleic acid sequencing technology.
  • the present invention relates to DNA tags and uses thereof, and more particularly, to DNA tags, oligonucleotides, DNA tag libraries, methods for their preparation, methods for determining DNA sample sequence information, and methods for DNA sequencing A method for a variety of DNA sample sequence information and a kit for constructing a DNA tag library. Background technique
  • Multiplex sequencing technology involves adding a DNA tag (also called Index) to identify the source of the sample between the linker sequence and the insert of each library, and using a tag-specific primer to mix multiple library samples. Sequencing is performed to determine the sequence of each library and its corresponding DNA tag, and then different libraries are distinguished based on each tag sequence to determine the DNA sequence of each library. Using multiplex sequencing technology, it is possible to achieve sequencing and sequencing of multiple library samples, thereby avoiding waste of sequencing resources.
  • a DNA tag also called Index
  • the current multiplex nucleic acid sequencing technology generally uses the same length of DNA tags to mix different libraries and simultaneously sequence them.
  • the detection insertion is often caused by the bias of the bases in the tags.
  • Fragment intensity parameters fluctuate, which affects the quality of the output data, resulting in unreliable data results, can not truly reflect the relevant information of the sample, and also leads to low repeatability of the experimental results.
  • the present invention at least solves one of the technical problems existing in the prior art.
  • the present invention provides a set of DNA tags (herein also simply referred to as "tags”) that can be used for multiplex nucleic acid sequencing and their applications.
  • the invention provides a set of isolated DNA tags.
  • the above DNA tag can be effectively applied to the current multiplex nucleic acid sequencing technology, that is, a DNA tag library of a plurality of samples can be simultaneously constructed by using the above DNA tag (in this case, sometimes referred to as a "tag library").
  • a DNA tag library of a plurality of samples can be simultaneously constructed by using the above DNA tag (in this case, sometimes referred to as a "tag library").
  • the DNA tag library derived from different samples can be mixed and sequenced, and the DNA sequence of the DNA tag library can be performed based on the DNA tag.
  • Classification which can obtain DNA sequence information of a variety of samples, thereby making full use of high-throughput sequencing technology, such as using Solexa sequencing technology, while sequencing multiple DNA tag libraries, thereby improving the sequencing efficiency of DNA tag libraries and Flux.
  • DNA tags of various samples can be constructed using DNA tags according to embodiments of the present invention, and multiplex nucleic acid sequencing can be performed to accurately perform a plurality of DNA tag libraries. Distinguish and effectively reduce data output bias, the resulting sequencing data is reliable, stable and repeatable.
  • the present invention provides a set of isolated oligonucleotides for introducing the above DNA tag into sample DNA or an equivalent thereof, thereby being applicable to multiplex nucleic acid sequencing technology.
  • a set of isolated oligonucleotides according to an embodiment of the invention having a first strand and a second strand, each of said strands being composed of a nucleotide represented by SEQ ID NO: (3N-1), respectively
  • these oligonucleotides respectively have a DNA tag according to an embodiment of the present invention as described above, and thus The corresponding DNA tag can be introduced into the DNA or its equivalent by a ligation reaction.
  • the first chain (also referred to herein as "sense sequence") and the second chain (also referred to herein as "antisense sequence”) are named IndexN adapter F and IndexN adapter R, respectively.
  • N 1 - 6 of any integer, the sequence of which is shown in Table 1 below (the sequence directions shown in the table are all 5' - 3' directions).
  • the corresponding DNA tag linker can be formed by subjecting the sense sequence IndexN adapter F and its corresponding antisense sequence IndexN adapter R to an equimolar annealing treatment.
  • the DNA tag is introduced into the DNA of the sample or its equivalent, whereby a DNA tag library having the DNA tag of the present invention can be constructed, thereby enabling accurate and efficient multiplex nucleic acid sequencing.
  • the inventors have surprisingly found that when constructing a DNA tag library containing various DNA tags using oligonucleotides having different tags for the same sample, the resulting sequencing data results are very stable and reproducible. .
  • the invention also provides a set of isolated PCR tag primers for introducing the above DNA tag into a sample DNA or the like.
  • a set of isolated PCR tag primers according to embodiments of the invention having a first strand and a second strand, wherein the first strand consists of the nucleotides set forth in SEQ ID NO: 39, respectively, and the nucleoside of the second strand ACGTGTGCTCTTCCGATCT, wherein XXXXX is one of a set of isolated DNA tags in accordance with an embodiment of the present invention.
  • the set of PCR tag primers respectively have the DNA tag according to the embodiment of the present invention as described above, and the PCR tag primer can be introduced into the DNA of the sample or its equivalent by PCR reaction using the PCR tag primer, thereby correspondingly
  • the DNA tag is introduced into the DNA or its equivalent, thereby obtaining a DNA tag library containing the corresponding DNA tag, thereby enabling the DNA tag library of the plurality of samples to be mixed and sequenced, that is, the PCR tag primer of the present invention can be effective Applied to multiplex nucleic acid sequencing technology.
  • the inventors have surprisingly found that when the DNA tag libraries containing various DNA tags are separately constructed using PCR tag primers having different tags for the same sample, the stability and reproducibility of the obtained sequencing data results are very high. it is good.
  • the present invention provides a method of constructing a DNA tag library.
  • the method comprises the steps of: fragmenting a DNA sample to obtain a DNA fragment; end-repairing the resulting DNA fragment to obtain a DNA fragment subjected to end repair; and performing the DNA fragment subjected to end repair A base A is added at the 3' end to obtain a DNA fragment in which the base A is added at the 3' end; a DNA fragment in which the base A is added at the 3' end is linked to a DNA tag linker to obtain a ligation product, wherein the DNA tag linker comprises a front One of a set of isolated DNA tags according to an embodiment of the present invention; amplifying the resulting ligation product to obtain an amplification product; and isolating and recovering the amplification product, the amplification product constituting a DNA tag library.
  • the method of constructing a DNA tag library according to an embodiment of the present invention can be effectively applied to a multiplex nucleic acid sequencing technology, and specifically, a method of constructing a DNA tag library according to an embodiment of the present invention can effectively perform an embodiment according to the present invention
  • the DNA tag is introduced into a DNA tag library constructed for the sample DNA, and the sequence information of the sample DNA and the sequence information of the DNA tag combination can be obtained by sequencing the DNA tag library, thereby distinguishing the source of the sample DNA.
  • the inventors have surprisingly found that different labels are used based on the above methods.
  • the constructed DNA tag library containing the DNA tag of the embodiment of the present invention is applied to the multiplex nucleic acid sequencing technology, the data output bias problem can be effectively reduced compared with the conventional multiplex nucleic acid sequencing technology, and the obtained sequencing data result is obtained. Reliable, stable and repeatable.
  • the present invention provides a DNA tag library.
  • the DNA tag library is obtained by a method of constructing a DNA tag library according to an embodiment of the present invention.
  • the inventors have found that the DNA tag library of the present invention can be effectively applied to a high-throughput sequencing platform, thereby efficiently obtaining accurate and reliable sequencing data, and the resulting sequencing data is stable and reproducible.
  • the invention provides a method of determining DNA sample sequence information.
  • the method comprises the steps of: constructing a DNA tag library of the DNA sample according to a method of preparing a DNA tag library according to an embodiment of the present invention; and sequencing the DNA tag library to determine the DNA sample Sequence information. Based on this method, the sequence information of the DNA sample in the DNA tag library and the sequence information of the DNA tag can be efficiently obtained, thereby enabling differentiation of the source of the DNA sample. Further, the inventors have surprisingly found that the use of the method according to an embodiment of the present invention to determine DNA sample sequence information can effectively reduce the problem of data output bias, and can accurately distinguish a plurality of DNA tag libraries.
  • the present invention also provides a method of determining a plurality of DNA sample sequence information.
  • the method comprises the steps of: constructing a DNA tag library of the DNA sample, independently of each of the plurality of samples, according to a method of constructing a DNA tag library according to an embodiment of the present invention, wherein a different DNA sample using DNA tags of different and known sequences; combining the DNA tag libraries of the plurality of samples to obtain a DNA tag library mixture; sequencing the DNA tag library mixture to obtain the Sequence information of the DNA sample and sequence information of the tag; and classifying sequence information of the DNA sample based on sequence information of the tag to determine DNA sequence information of the plurality of samples.
  • the method according to an embodiment of the present invention can make full use of high-throughput sequencing technology, for example, using Solexa sequencing technology, and simultaneously sequencing a DNA tag library of a plurality of samples, thereby improving the efficiency of DNA tag library sequencing and communication.
  • the quantity can simultaneously improve the efficiency of determining the sequence information of a plurality of DNA samples, and can effectively reduce the problem of data output bias compared with the conventional multiplex nucleic acid sequencing technology, and the sequencing data is more accurate and reliable.
  • the present invention provides a kit for constructing a DNA tag library.
  • a DNA tag according to an embodiment of the present invention can be conveniently introduced into a constructed DNA tag library.
  • the kit according to an embodiment of the present invention can be effectively applied to a multiplex nucleic acid sequencing technique.
  • the invention proposes the use of a set of different length, preferably gradient length tags for the construction and/or sequencing of a sequencing tag library, wherein the tag is an oligonucleotide sequence, preferably Is a nucleotide sequence of 2 - lOObp.
  • the tag is included in a PCR primer for amplifying a sequence of interest
  • the corresponding corresponding PCR primers are constructed and introduced into the sequence to be sequenced by PCR, and the PCR tag primer is used as a 5' primer of PCR, or a 3' primer, or a 5' primer and a 3' primer which are simultaneously used as PCR. .
  • the tag is embedded in a PCR primer for amplifying a sequence of interest, or with or without a linker and a PCR primer for amplifying a sequence of interest.
  • the 5' ends or the 3' ends are ligated to form respective corresponding tag PCR primers.
  • the tag is included in a linker of a tag library to form a corresponding tag link, the tag linker being used as a 5' linker, 3' linker of the tag library, or both As the 5' linker and 3' linker of the tag library.
  • the tag is inserted into the connector, or is connected to the end of the connector with or without a connector, preferably not connected to the end of the connector, thereby forming a self-corresponding tag connector.
  • the tag constitutes a tag PCR primer and a tag linker, and is used for the construction and/or sequencing of a sequence tag library.
  • the invention proposes a set of tags of different lengths, preferably gradient lengths, for use in the construction and/or sequencing of sequencing tag libraries.
  • the present invention proposes a set of gradient labels comprising or consisting of: 6 gradient labels shown in Table 1 or gradient labels having a base difference of 1 base At least 2, or at least 3, or at least 4, or at least 5, or all 6,
  • the set of gradient labels preferably includes at least Indexl and Index2, or Index3 and Index4, or Index5 and Index6 of the six gradient labels shown in Table 1, or a combination of any two or more of them.
  • the one base difference comprises one base substitution, addition or deletion in the sequence of the six gradient tags shown in Table 1.
  • the invention proposes the use of the gradient label described above for sequencing a library of tags, in particular the construction and/or sequencing of an Illumina/Solexa sequencing tag library, wherein the gradient tag is included for
  • the sequencing tag library in particular the 5' end of linker 2 of the IUumina/Solexa sequencing tag library, constitutes the corresponding corresponding gradient tag linker 2, which serves as a sequencing tag library, in particular 3' of the Illumina/Solexa sequencing tag library Connector.
  • the gradient label is contained in the 5' end of the joint 2, including the gradient label being connected to the end 5 of the joint 2 with or without a linker, or inserted into the 5' end of the joint 2 Preferably, it is not connected to the 5' end of the linker 2 by a linker.
  • the present invention proposes a sequencing tag library constructed using the aforementioned method, in particular
  • the invention proposes a set of gradient tag binders 2 comprising the gradient labels described above, comprising the gradient tags of claim 1 at the 5' end, and preferably for use as a sequencing tag library
  • the 3' linker of the Illumina/Solexa sequencing tag library the set of gradient tag linkers 2 comprising or consisting of: 6 gradient tag linkers 2 shown in Table 1 or differing from the gradient tag sequences contained therein 1 At least 2, or at least 3, or at least 4, or at least 5, or all 6 of the base-based joints
  • the set of gradient label joints 2 preferably includes at least Indexl adapter2 F/R and Index2 adapter2 F/R, or Index3 adapter2 F/R and Index4 adapter2 F/R of the six gradient label joints 2 shown in Table 1, Or Index5 adapter2 F/R and Index6 adapter2 F/R, or a combination of any two or more of them.
  • the invention proposes a gradient tag adaptor 2, wherein said one base difference comprises a substitution, addition or deletion of one base in the gradient tag sequence.
  • the invention proposes the use of a gradient tag adaptor 2 for sequencing a library of libraries, in particular an Illumina/Solexa sequencing tag library, for use as a sequencing tag library,
  • a gradient tag adaptor 2 for sequencing a library of libraries, in particular an Illumina/Solexa sequencing tag library, for use as a sequencing tag library,
  • the 3' linker of the Illumina/Solexa sequencing tag library is particularly advantageous.
  • the invention proposes a library of sequencing tags constructed using the gradient tag linker 2, in particular an Illumina/Solexa sequencing tag library, wherein the gradient tag linker 2 is used as a library of sequencing tags, in particular Illumina /Solexa sequencing tag library 3' linker.
  • the present invention provides a method of constructing a library of sequencing tags, in particular an Illumina/Solexa sequencing tag library, characterized in that a set of tags having labels of different lengths, preferably gradient lengths, is used.
  • a sequencing tag library used as a sequencing tag library, in particular a 3' linker for the Illumina/Solexa sequencing tag library.
  • a method of constructing a library of sequencing tags comprises:
  • Breaking DNA Breaking DNA by mechanical means to produce DNA fragments with sticky ends, including but not limited to Bioruptor, Hydroshear and Covaris;
  • End repair fill the sticky end of the DNA fragment by a ligation reaction
  • the method is characterized in that a different gradient linker 2 selected from Table 1 or a linker differing from the gradient tag sequence contained therein by 1 base is used as a sequencing tag library, in particular Illumina /Solexa sequencing tag library 3' linker.
  • a method of constructing a library of sequencing tags comprises:
  • n being an integer and 1 ⁇ 6, preferably 2 ⁇ 6, the genomic DNA sample being from any eukaryotic sample, including but not limited to human genomic DNA samples;
  • Breaking DNA Breaking DNA by mechanical means to produce DNA fragments with sticky ends, including but not limited to Bioruptor, Hydroshear, and Covaris;
  • End repair fill the sticky end of the DNA fragment by a ligation reaction
  • the linker 1 comprises the linker: 5 '-TACACTCTTTCCCTACACG ACGCTCTTCCGATCTATCACT (SEQ ID NO: 37) and 5, - /GTGATAGATCGGAAGAGCACAC GTCTGAACTCCAGTCAC (SEQ ID NO: 38).
  • the gradient label joint 2 comprises at least 2 of the 6 gradient label joints 2 shown in Table 1 or a linker which differs by 1 base from the gradient label sequence contained therein, or at least 3 , or at least 4, or at least 5, or all 6,
  • the set of gradient label joints 2 preferably includes at least Indexl adapter2 F/R and Index2 adapter2 F/, or Index3 adapter2 F/R and Index4 adapter2 F/R of the six gradient label joints 2 shown in Table 1, or Index5 adapter2 F/R and Index6 adapter2 F/R, or a combination of any two or more of them.
  • the one base difference comprises one base substitution, addition or deletion in the gradient tag sequence.
  • PCR in step 6 uses the following PCR primers:
  • the recovery of the library of the desired fragment in step 6) is carried out by agarose gel electrophoresis and gel recovery.
  • the sequencing method used in sequencing using the sequencing technique includes when the DNA is constructed
  • AC ACTCTTTCCCTAC ACGACGCTCTTC CGATCT.
  • the present invention also proposes a sequencing tag library constructed according to the foregoing method, in particular
  • Figure 1 is a schematic flow chart showing a method of constructing a DNA tag library according to an embodiment of the present invention
  • 2 is a schematic diagram showing the sequence of sequence reading of each sequencing reaction and the composition of the read sequence when sequencing a DNA tag library using the Illumina/Solexa sequencing platform according to an embodiment of the present invention
  • Figure 3 shows the results of comparison of the mass values of the first 10 Illumina/Solexa sequencing cycles of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein
  • Figure 3A shows the mass values of the first 10 Illumina/Solexa sequencing cycles of the DNA tag library of the invention of Example 1.
  • Figure 3B shows the mass values of the top 10 Illumina/Solexa sequencing cycles of the DNA tag library of the control
  • Figure 4 is a graph showing the comparison of the light intensities of the respective Illumina/So lexa sequencing cycles of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein
  • Figure 4A shows the average light intensity signal of each IUumina/Solexa sequencing cycle of the DNA tag library of the present invention of Example 1.
  • Figure 4B shows the average of the light intensity signals for each Illumina/Solexa sequencing cycle of the DNA tag library of the control
  • Figure 5 is a graph showing the results of comparison of base distributions of the Illumina/Solexa sequencing cycle of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein
  • Figure 5A is a graph showing the percentage distribution of bases of the Illumina/Solexa sequencing cycle of the DNA tag library of the present invention of Example 1.
  • Figure 5B is a graph showing the percentage distribution of bases of the Illumina/Solexa sequencing cycle of the DNA tag library of the control
  • Figure 6 is a graph showing the comparison of the error rates of the Illumina/Solexa sequencing cycles of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein
  • Figure 6A is a graph showing the error rate of the Illumina/Solexa sequencing cycle of the DNA tag library of the present invention of Example 1.
  • Figure 6B Error rate plot showing the Illumina/So lexa sequencing cycle of the DNA tag library of the control.
  • the invention provides a set of isolated DNA tags.
  • DNA as used in the present invention may be any polymer comprising deoxyribonucleotides, including but not limited to modified or unmodified DNA.
  • a DNA tag according to an embodiment of the present invention, a DNA tag library having a tag is obtained by linking the DNA tag to the DNA of the sample or its equivalent, and the sequence of the sample DNA and the sequence of the tag can be obtained by sequencing the DNA tag library. Further, based on the sequence of the tag, the sample source of the DNA can be accurately characterized.
  • a DNA tag library of a plurality of samples can be simultaneously constructed, and the DNA sequence of the sample can be classified based on the DNA tag by mixing and simultaneously sequencing the DNA tag library derived from different samples.
  • DNA tag attached to the DNA of the sample or its equivalent should be understood broadly, including the DNA tag can be directly linked to the DNA of the sample to construct a DNA tag library, and can also be associated with the DNA of the sample.
  • a nucleic acid of the same sequence for example, may be the corresponding RA sequence or cDNA sequence, which has the same sequence as the DNA).
  • the inventors of the present application found that: In the present invention, in order to design an effective DNA tag, it is first necessary to consider the degree of sequence difference between the tag sequences and the base recognition rate. Second, in the case of a label mix of less than 6 samples, the GT content of each base site on the mixed label must be considered. Because the excitation fluorescence of thiol G and T is the same in the Solexa sequencing process, the excitation light of bases A and C is the same, so the "balance" of the base “GT” content and the base “AC” content must be considered. The base base “GT” content is 50%, which ensures the highest label recognition rate and the lowest error rate. Finally, consider the repeatability and accuracy of the data output.
  • a set of DNA tags In order to achieve efficient construction of the DNA tag library and sequencing, a set of DNA tags must be constructed to ensure reliable results and high reproducibility. The same DNA sample ensures that a library of DNA tags constructed using different tags in the set of DNA tags will result in consistent sequencing results, thus ensuring reliable and reproducible results. In addition, it is also necessary to avoid the appearance of 3 or more consecutive bases in the tag sequence, because 3 or more consecutive bases increase the error rate of the sequence during synthesis or sequencing, and also Try to avoid the DNA tag linker and the PCR tag primer itself forming a hairpin structure.
  • the inventors of the present application performed a large number of screening work, and selected a set of isolated DNA tags according to an embodiment of the present invention, which are respectively represented by the nucleotides represented by SEQ ID NO: (3N-2)
  • the sequence is as shown in Table 1 above, and will not be described again.
  • the set of isolated DNA tags is a gradient tag. Specifically, Indexl is 6 bp, Index 2 is 7 bp, Index 1 is 8 bp, Indexl-3 has a gradient of lbp increment, and similarly, Index 4 is 6 bp.
  • Index5 is 7bp
  • Index6 is 8bp
  • Index4-6 has a gradient of lbp increment, which can effectively reduce the bias of bases in the tag, and then effectively reduce these base tags when applied to multiplex nucleic acid sequencing technology.
  • the fluctuation of the light intensity parameters during the sequencing process can significantly improve the quality of the output data, and further, can significantly improve the reliability, stability and repeatability of the sequencing data.
  • the difference between each DNA tag is 5 bases or more. When any one of the bases of the DNA tag has a sequencing error or a synthetic error, the final recognition of the tag is not affected.
  • These tags can be applied to the construction of any DNA tag library. Built. There are currently no reports on the construction of these tags for DNA sample sequencing and sequencing by Solexa technology.
  • the set of DNA tags used consists of: at least 2, or at least 3, or at least 3 of the DNA tags shown in Table 1 or a DNA tag 1 base different therefrom 4, or at least 5, or all 6.
  • the set of DNA tags preferably includes at least Index 1 and Index 2, or Index 3 and Index 4, or Index 5 and Index 6 of the six DNA tags shown in Table 1, or any two of them. Combination of one or more.
  • the one base difference comprises one base substitution, addition or deletion in the sequence of the six DNA tags shown in Table 1.
  • the present invention also provides the use of a DNA tag according to an embodiment of the present invention in a multiplex nucleic acid sequencing technique, in particular, the use of a DNA tag in the construction and sequencing of a DNA tag library.
  • the DNA tag of the present invention can be contained in a linker of a DNA tag library to constitute a corresponding DNA tag linker, which is used as a 5' linker of a DNA tag library, a 3' linker, or both The 5' linker and the 3' linker of the DNA tag library, whereby the DNA tag can be introduced into the DNA tag library by a ligation reaction.
  • the DNA tag is inserted into the end of the DNA tag linker, or is ligated to the end of the DNA linker with or without a linker, preferably without the linker in the end of the DNA tag linker, thereby constituting the respective phase Corresponding DNA tag linker.
  • the DNA tag of the present invention can be contained in a PCR primer for amplifying a sequence of interest to constitute a corresponding PCR tag primer, which is used as a 5' primer of a PCR reaction, or a 3' primer, Alternatively, it can be used as a 5' primer and a 3' primer of a PCR reaction, whereby a DNA tag can be introduced into a DNA tag library by a PCR method.
  • the DNA tag is embedded in the PCR tag primer, or is linked to the 5' end or the 3' end of the PCR primer for amplifying the sequence of interest by or without a linker, thereby constituting the corresponding PCR tag. Primer.
  • the present invention provides the use of the aforementioned DNA tag for the construction and/or sequencing of a DNA tag library, particularly an Illumina/Solexa sequencing DNA tag library.
  • the DNA tag is contained in a DNA tag library, particularly the 5' end of the 3' linker of the Illumina/Solexa sequencing DNA tag library, thereby constituting the corresponding DNA tag linker, which is used as DNA tag library, specifically the 3, linker of the Illumina/Solexa sequencing DNA tag library.
  • the present invention provides the use of the aforementioned DNA tag for the construction and/or sequencing of a DNA tag library, particularly an Illumina/Solexa sequencing DNA tag library.
  • the DNA tag is contained in a DNA tag library, particularly the 5' end of the 3' linker of the IUumina/Solexa sequencing DNA tag library, wherein the DNA tag is ligated to the 5' end of the 3' linker with or without a linker, or is inserted In the 5' end of the 3' linker.
  • the linker is a sequence of 1 - 10 bases, preferably a sequence of 1 - 5 bases, more preferably a sequence of 1 - 3 bases.
  • the invention provides a set of isolated oligonucleotides that can be used to introduce a DNA tag as described above into the DNA of a sample, thereby constructing a library of DNA tags.
  • the corresponding oligonucleotides can be formed by annealing the first strand and the second strand constituting the corresponding oligonucleotide, respectively.
  • the above oligonucleotides each have a DNA tag according to an embodiment of the present invention as described above, and thus the corresponding DNA tag can be introduced into the DNA of the sample or its equivalent by a ligation reaction.
  • the sequences of these oligonucleotides are as shown in Table 1 above, and are not described herein again.
  • the set of DNA tag linkers which comprise a DNA tag of the invention at the 5' end, and preferably serve as a DNA tag library, in particular a 3' linker of an Illumina/Solexa sequencing DNA tag library .
  • the set of DNA tag linkers comprises or consists of: 6 DNA tag linkers shown in Table 1 or at least 2 of the DNA tag linkers differing from the DNA tag sequence contained therein by 1 mechanical group , or at least 3, or at least 4, or at least 5, or all 6.
  • these DNA tag linkers preferably include at least Indexl adapter F/R and Index2 adapter F/R, or Index3 adapter F/R and Index4 adapter F in the six DNA tag headers 2 shown in Table 1. /R, or Index5 adapter F/R and Index6 adapter F/R, or a combination of any two or more of them.
  • the one base difference in the DNA tag linker described above includes one base substitution, addition or deletion in the sequence of the six DNA tags shown in Table 1.
  • the invention provides the use of a DNA tag linker for the construction and/or sequencing of a DNA tag library, in particular an Illumina/Solexa sequencing DNA tag library, which is used as a DNA tag library, in particular Is the 3' linker of the Illumina/Solexa sequencing DNA tag library.
  • the present invention provides a set of isolated PCR tag primers which can be used to introduce a DNA tag as described above into the DNA of a sample, thereby constructing a DNA tag library.
  • the set of isolated PCR tag primers has a first strand and a second strand, wherein the first strand is composed of the nucleotides represented by SEQ ID NO: 39, and the second strand is nucleoside
  • the acid sequence is as follows: CAAGCAGAAGACGGCATACGAGATXXX XXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, wherein XXXXX is one of a group of isolated DNA tags according to an embodiment of the present invention.
  • the PCR tag primers of the present invention each have a DNA tag according to an embodiment of the present invention as described above, and a PCR tag primer can be introduced into a sample DNA or an equivalent thereof by a PCR reaction using a PCR tag primer.
  • the corresponding DNA tag is introduced into DN A or its equivalent, thereby enabling introduction of the DNA tag into the DNA tag library, thereby enabling sequencing of multiple samples by performing hybrid sequencing of the DNA tag library of each sample. And can effectively reduce the problem of data output bias, the obtained sequencing data is reliable, stable, and repeatable.
  • the PC of the present invention The R-tag primer can be used as a 5' primer or a 3' primer for PCR reaction, or as both a 5' primer and a 3' primer for PCR reaction, thereby enabling introduction of a DNA tag into a DNA tag library by PCR. .
  • a DNA tag library constructed using the above DNA tag linker and PCR tag primer is also provided.
  • the present invention also provides a method of constructing a DNA tag library using the above DNA tag linker.
  • the method may include:
  • a DNA sample is fragmented to obtain a DNA fragment.
  • the method of fragmenting a DNA sample is not particularly limited.
  • the fragmentation is carried out by at least one selected from the group consisting of an atomization method, an enzyme digestion method, and an ultrasonication method.
  • the DNA sample is fragmented using ultrasonic disruption.
  • the DNA sample is fragmented using a Covaris interrupter.
  • the obtained DNA fragment can be about 500 bp in length, whereby the efficiency of constructing a DNA tag library and subsequent sequencing can be further improved.
  • the source of the DNA sample is not particularly limited.
  • the DNA sample may be derived from any common biological sample, such as a plant, animal or microorganism.
  • the DNA sample may be derived from at least one selected from the group consisting of Arabidopsis thaliana, rice, human, mouse, and Escherichia coli.
  • a DNA tag library of a plurality of common model organisms can be efficiently constructed.
  • the DNA sample may be a human DNA sample, and more specifically, may be a human genomic DNA sample.
  • the DNA fragment is end-repaired to obtain a DNA fragment that has been repaired at the end.
  • the method of performing end repair of the DNA fragment is not particularly limited.
  • the end repair is carried out using a Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragment has 5 ' ⁇ 3 'polymerase activity and 3 ' ⁇ 5 ' exonuclease Active, but lacking 5 ' ⁇ 3 ' exonuclease activity.
  • the terminal-repaired DNA fragment is subjected to the base A at the 3' end to obtain a DNA fragment in which the base A is added at the 3' end.
  • the method of adding the base A at the 3' end of the DNA fragment which has been subjected to end repair is not particularly limited.
  • Klenow (3 '-5' exo-) can be used to carry out the end-repaired DNA fragment 3, and add base A.
  • bases are added to the 3' ends of the two oligonucleotide strands of the end-repaired DNA fragment.
  • a DNA fragment in which the base A is added at the 3' end is ligated to a DNA tag linker to obtain a ligation product, wherein the DNA tag linker comprises one of a group of isolated DNA tags according to an embodiment of the present invention.
  • a method of attaching a DNA fragment in which the base A is added to the DNA tag linker at the 3' end is not particularly limited.
  • the DNA fragment in which the base A is added to the 3' end and the DNA tag linker is carried out using T4 DNA ligase.
  • the DNA tag linker may be one selected from the isolated oligonucleotides according to the embodiments of the present invention as described above. Then, the resulting ligation product is amplified to obtain an amplification product. According to an embodiment of the invention, prior to the amplification of the ligation product, the step of fragment selection of the ligation product is further included.
  • fragment selection can be performed using 2% agarose gel electrophoresis.
  • the length of the ligation product selected by the fragment may be about 620 bp.
  • the ligation product is subjected to amplification: by a PCR reaction using primers having nucleotide sequences as shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
  • the obtained amplification product is separated and recovered, and the amplification product constitutes a DNA tag library.
  • the method for isolating and recovering the amplified product is also not particularly limited, and those skilled in the art can select an appropriate method and apparatus for separation according to the characteristics of the amplified product, for example, by electrophoresis and recovering a PCR of a specific length.
  • the method of amplifying the product is recovered.
  • the amplification product may be isolated by at least one selected from the group consisting of agarose gel electrophoresis, magnetic bead purification, and purification column purification.
  • the isolated amplified product may be about 620 bp in length.
  • a DNA tag according to an embodiment of the present invention can be efficiently introduced into a DNA tag library constructed for a DNA sample.
  • the DNA tag library can be sequenced to obtain sequence information of the DNA sample and sequence information of the DNA tag, thereby distinguishing the source of the DNA sample.
  • the method for constructing a DNA tag library provided by the invention can fully utilize a high-throughput sequencing platform, meets the requirements of high-throughput sequencing, saves sequencing resources, thereby reducing the cost of sequencing, and can effectively be compared with the conventional multiplex nucleic acid sequencing technology. To reduce the bias of data output, the obtained sequencing data is reliable, stable, and reproducible.
  • the method of constructing a DNA tag library of the present invention may include:
  • genomic DNA sample can be from any eukaryotic sample, including but not limited to human genomic DNA samples;
  • Linkers are ligated to obtain a ligation product, wherein the DNA tag linker comprises a set of isolated DNA tags according to embodiments of the invention
  • the method for constructing a DNA tag library of the present invention may further comprise: 1) providing n genomic DNA samples, n being an integer and 1 ⁇ n ⁇ 6, preferably 2 ⁇ n ⁇ 6, wherein the genomic DNA sample can be from any eukaryotic sample, including but not limited to human genomic DNA samples;
  • the linker of the Illumina/Solexa sequencing library used in the method for constructing a DNA tag library of the present invention described above is a linker 1 whose sequence is as follows:
  • the DNA tag linker used in the above method for constructing a DNA tag library of the present invention comprises or consists of: 6 DNA tag linkers shown in Table 1 or a DNA tag sequence contained therein is different from one At least two, or at least three, or at least four, or at least five, or all six of the base DNA tag linkers.
  • these DNA tag linkers preferably include at least Index 1 adapter F/R and Index 2 adapter F/R, or Index 3 adapter F/R and Index 4 adapter in the 6 DNA tag connectors 2 shown in Table 1.
  • the one base difference in the DNA tag linker described above includes a substitution, addition or deletion of one of the sequences of the six DNA tags shown in Table 1.
  • the PCR reaction in the above method for constructing a DNA tag library of the present invention employs the following PCR primers:
  • the method of constructing a DNA tag library of the present invention may further comprise:
  • a set of DNA tags of different lengths of the present invention may be separately contained in PCR primers for amplifying a sequence of interest, thereby constituting respective corresponding PCR tag primers, thereby, the present invention
  • the method for constructing the DNA tag library may include: firstly, the total DNA sample is disrupted into a DNA fragment of a certain length by mechanical method or enzymatic cleavage, and after the DNA fragment is ligated to the linker, the PCR-containing primer pair contains the DNA fragment ( That is, the ligation product of the above-mentioned target sequence is amplified, and finally the amplified product is recovered by agarose electrophoresis and gel-cutting, thereby obtaining a DNA tag library composed of the amplified product.
  • a set of DNA tags of different lengths of the present invention can be respectively embedded in a linker (e.g., a terminal) of an existing library to constitute a corresponding DNA tag linker, thereby constructing the present invention.
  • the method of the DNA tag library may include: firstly, the total DNA sample is disrupted into a length of the DNA fragment by mechanical or enzymatic cleavage, and after the DNA fragment is end-repaired and a random sticky end is formed, the DNA tag is combined with the DNA tag.
  • the linker is ligated to obtain a ligation product, and then the ligation product containing the DNA fragment (ie, the sequence of interest described above) is amplified by a specific PCR primer, and finally the amplified product is recovered by agarose electrophoresis and gel-cutting.
  • a DNA tag library consisting of amplification products.
  • a set of DNA tags of different lengths of the present invention may be separately contained in PCR primers for amplifying a sequence of interest, thereby constituting respective corresponding PCR tag primers, and at the same time one of the present invention
  • the DNA tags of different lengths are respectively inserted into the adaptors (for example, the ends) of the existing library, thereby constituting the corresponding DNA tag linkers.
  • the method for constructing the DNA tag library of the present invention may include: first using the total DNA sample Mechanically or enzymatically interrupting a DNA fragment of a certain length, and then ligating the DNA fragment to a DNA tag linker to obtain a ligation product, and then using a PCR tag primer pair containing the DNA fragment (ie, the sequence of interest described above) The ligation product is extensively increased, and finally the product is recovered by agarose electrophoresis and gel-cutting to obtain a DNA tag library composed of the amplified product.
  • a method for constructing a DNA tag library particularly an Illumina/Solexa sequencing DNA tag library, using the DNA tag linker of the present invention shown in Table 1 is used as a DNA tag library, in particular Is the linker of the Illumina/Solexa sequencing DNA tag library.
  • the present invention also provides a kit for constructing a DNA tag library.
  • a DNA tag according to an embodiment of the present invention can be conveniently introduced into a constructed DNA tag library.
  • a DNA tag library can also be included in the kit, and details are not described herein.
  • the present invention also provides a DNA tag library which is constructed in accordance with the present invention
  • the DNA tag library having the DNA tag according to an embodiment of the present invention may Effectively applied to high-throughput sequencing technologies such as Solexa technology, the obtained nucleic acid sequence information such as DNA sequence information can be accurately classified by sample source by obtaining a tag sequence, and the obtained sequencing data is accurate and reliable. Good stability and repeatability.
  • the DNA tag library of the invention employs a DNA tag linker as a DNA tag library, in particular a 3' linker of the IUumina/Solexa sequencing tag library.
  • the present invention also provides a method of determining DNA sample sequence information.
  • the method comprises: constructing a DNA tag library according to a method for constructing a DNA tag library according to an embodiment of the present invention; and then, sequencing the constructed DNA tag library to determine sequence information of the DNA sample. Based on this method, the sequence information of the DNA sample in the DNA tag library and the sequence information of the DNA tag can be efficiently obtained, thereby distinguishing the source of the DNA sample.
  • the inventors have surprisingly found that the use of a method according to an embodiment of the present invention to determine DNA sample sequence information can effectively reduce the problem of data output bias, and can accurately distinguish a plurality of DNA tag libraries, and obtain sequencing.
  • the constructed DNA tag library can be sequenced by any known method, and the type thereof is not particularly limited.
  • DNA tag libraries can be sequenced using Solexa sequencing technology.
  • suitable sequencing primers can be selected for sequencing according to specific conditions.
  • the present invention provides a method of determining sequence information for a plurality of DNA samples.
  • the method comprises the steps of: constructing a DNA tag library of the DNA sample according to the method of constructing the DNA tag library of the embodiment of the present invention, respectively, for each of the plurality of samples, wherein Different DNA samples use DNA tags of different and known sequences; DNA library libraries of various samples are combined to obtain a DNA tag library mixture; DNA tag library mixture is sequenced to obtain sequence information of the DNA sample and Sequence information of the label combination; and sorting the sequence information of the DNA sample based on the sequence information of the label combination to determine DNA sequence information of the plurality of samples.
  • the term "various" is used in at least two.
  • the method of sequencing the DNA tag library mixture is not particularly limited.
  • the obtained DNA tag library mixture can be sequenced using Solexa sequencing technology to obtain sequence information of the DNA sample and sequence information of the tag.
  • the method according to an embodiment of the present invention can make full use of high-throughput sequencing technology, for example, using Solexa sequencing technology, and simultaneously sequencing DNA libraries of various samples, thereby improving the efficiency and throughput of DNA library sequencing. And can effectively reduce the problem of data output bias, the obtained sequencing data is accurate, reliable, stable and repeatable.
  • 1 to 2 ⁇ ⁇ human peripheral blood genomic DNA samples were taken for sample detection. Specifically, the concentration of the DNA sample, the ratio of OD260/280, and the ratio of OD260/230 were measured using NanoDrop 1000, and the integrity of the sample was detected by agarose gel electrophoresis, and after the test was passed, it was used.
  • Sample concentration The concentration of the sample should not be lower than 100 ng/ ⁇ ; Sample purity: OD260/280 ratio should be between 1.8 and 2.0, no protein, polysaccharide and RA contamination; sample integrity: electrophoresis results show that DNA samples should not be degraded;
  • the total sample amount is required to be not less than 45 ⁇ ⁇ :
  • the above-tested DNA sample was fragmented to obtain a DNA fragment.
  • there are two methods for fragmenting DNA samples namely Nebulization and Covaris, which can break the DNA of the sample to a range of 100-800 b and the main band is about 500 bp (if If the sample is interrupted DNA, you can skip this step).
  • the DNA sample was fragmented by atomization, and the obtained DNA fragment was 100-800 bp in length and 500 bp in the main band.
  • the conventional label joint mixture was obtained by combining the conventional label joints formed by annealing the corresponding sense sequence PE IndexN adapter F and the antisense sequence PE IndexN adapter R shown in Table 2 above.
  • the PCR reaction uses the following PCR primers:
  • the amplified product was electrophoresed on a 2% agarose gel at 100 V for 120 min, and then 620 bp (the length of the cut fragment can be calculated by n+120 bp, where n insert size) was used, and then used.
  • the QIAquick Gel Extraction Kit (Qiagen) recovers the cut pieces to obtain a DNA tag library, which is then dissolved in 40 ⁇ l of elution buffer for later use.
  • a DNA tag library was constructed using the Multiplexing Sample Preparation Oligonucleotide Kit (PE-400-1001), using the standard protocol of the kit, using the six DNA tags according to the examples of the present invention in Table 1 and their corresponding DNA tag adapters.
  • the specific steps are the same as the control examples, where the difference is:
  • Step 5 using a DNA tag adapter mixture (Index Adapter Oligo mix) instead of a conventional label linker mixture (PE Index Adapter Oligo Mix), wherein the DNA tag linker mixture is obtained by using the sense sequence IndexN adapter F and the counter shown in Table 1.
  • a DNA tag linker formed by annealing the index sequence IndexN adapter R is obtained by combination.
  • Step 7 The procedure for the PCR reaction is:
  • the DNA tag libraries constructed in the Comparative Example and Example 1 were sequenced using a HiSeq2000 sequencer in strict accordance with the recommended procedure of the instrument to obtain sequencing data.
  • the sequencing primers used are:
  • Figure 2 shows a sequence of sequence reads and their sequence composition for each Reads when sequencing a DNA tag library using the Solexa sequencing platform. As shown in Figure 2, where Readl represents the sequence detected by Sequencing Reaction 1, Read 1 Seq Primer represents the sequencing primer used.
  • Data processing software includes, but is not limited to, HiSeq Control Software (HCS), Pipeline. CASAVA, SOAP, and ELAND.
  • FIG. 3 shows the results of comparison of the mass values (Q20) of the first 10 sequencing cycles of the DNA tag library of the present invention of Example 1 and the DNA tag library of the comparative example, wherein A shows the invention of the examples.
  • the mass value of the first 10 sequencing cycles of the DNA tag library B shows the mass values of the first 10 sequencing cycles of the DNA tag library of the control.
  • the abscissa indicates the number of cycles
  • the ordinate indicates the quality.
  • the quality value (Q-Vahie) can reflect the quality of sequencing, and it is between 0-40. Within this range, the higher the value, the better the quality.
  • Q20 refers to the proportion of bases with mass values greater than 20 in all bases, which can reflect the quality of the sequence sequenced.
  • the DNA tag of the present invention is constructed by embedding a DNA tag of a gradient length change of the present invention into a linker, constructing a DNA tag library of the present invention, and sequencing the DNA tag library using the Illumina/Solexa technique.
  • the mass value Q20 of the first 10 sequencing cycles of the library was maintained at 0.9; a 6 bp fixed-length conventional tag was inserted into the adaptor to construct a DNA library of the control, and the DNA tag library was sequenced using the Illumina/Solexa technique.
  • Fig. 4-6 shows the results of comparison of changes in light intensity, base distribution, and error rate with the number of cycles of the DNA library of the present invention in Example 1 and the tag library of the control.
  • 4 shows the results of comparison of the light intensities of the respective sequencing cycles of the DNA tag library of Example 1 and the tag library of the control example, wherein A shows the light intensity of each sequencing cycle of the DNA tag library of the present invention of Example 1.
  • the signal average, B shows the average of the light intensity signals for each round of sequencing cycles of the DNA tag library of the control.
  • the abscissa indicates the number of cycles, and the ordinate indicates the signal intensity average (Sign Mean).
  • FIG. 5 shows the results of comparison of base distributions along the Reads position of Solexa sequencing data of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein A shows the inventive example of Example 1.
  • the abscissa indicates the position along Reads
  • the ordinate indicates the percentage of each base at the position of the Reads
  • the graph shows the various base ratios measured in each sequencing.
  • FIG. 6 shows the results of comparison of error rates along the Reads position of Solexa sequencing data of the DNA tag library of the present invention and the DNA tag library of the control according to an embodiment of the present invention, wherein A shows the DNA of the present invention of Example 1.
  • the error rate map of the Solexa sequencing data of the tag library along the Reads position B shows the DNA tag library of the control example
  • the error rate graph of Solexa sequencing data along the Reads location As shown in Fig. 6, the abscissa indicates the position along Reads, the ordinate indicates the error rate (ie, the proportion of the sequencing error occurs at the position of the Reads), and the solid line indicates the error rate (ie, at the position of the Reads, the sequencing error occurs).
  • the ratio of the base line indicates the proportion of bases that cannot be analyzed.
  • the figure shows the difference in error rate between different libraries.
  • the DNA tag library of the present invention of Example 1 has no significant difference from the tag library of the control sample from the three parameters of light intensity, base distribution, and error rate as a function of the number of cycles. . It is shown that the DNA tag library constructed using the DNA tag of the gradient length variation of the present invention and the DNA tag library constructed using the conventional tag of the fixed length are not significantly different as a whole, and the use of the DNA tag of the present invention does not affect the DNA.
  • the overall sequencing results of the tag library, and the sequencing of the transition from the gradient tag to the insert can significantly improve the quality of the base in excess.
  • the DNA tag of the present invention is illustrated to be superior to conventional tags in conventional multiplex nucleic acid sequencing techniques.
  • the tag library can increase 83.5 M of data when running a HiSeq sequencer and can increase the availability of data.
  • the DNA tag library kit can be effectively applied to the construction and sequencing of DNA sequencing libraries of sample DNA, and can effectively reduce the problem of data output bias, and the sequencing results are accurate, reliable, stable and reproducible.

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Abstract

L'invention concerne une étiquette à ADN, un adaptateur d'étiquette à ADN et une amorce d'étiquette pour PCR pour la construction d'une banque d'étiquettes à ADN. L'invention concerne également une banque d'étiquettes à ADN et un procédé et une trousse pour la construction de ladite banque. L'invention concerne également un procédé de détermination d'informations de séquence pour un ou plusieurs échantillons d'ADN.
PCT/CN2012/071893 2011-03-03 2012-03-02 Étiquette à adn et son utilisation Ceased WO2012116661A1 (fr)

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