WO2015196752A1

WO2015196752A1 - A method and a kit for quickly constructing a plasma dna sequencing library

Info

Publication number: WO2015196752A1
Application number: PCT/CN2014/093698
Authority: WO
Inventors: Di Chen; Yali Li; Tiancheng Li; Yanbin SHI; Jianghua ZHANG; Kai Wu
Original assignee: Berry Genomics Co Ltd
Current assignee: Berry Genomics Co Ltd
Priority date: 2014-06-24
Filing date: 2014-12-12
Publication date: 2015-12-30
Anticipated expiration: 2016-12-24
Also published as: CN105296602B; CN111748606A; HK1220994A1; CN105296602A

Abstract

The present invention provides a method for constructing a plasma DNA sequencing library, comprising: extracting plasma DNA, optionally end-filling the plasma DNA, adding A on the 3'end of the plasma DNA, connecting the plasma DNA with a sequencing linker, and purifying the connected product. The three steps of end-filling, adding A and connecting the plasma DNA with a linker can be completed in one reaction system. The use of PCR is not necessary for constructing the library. The present invention also provides a kit for constructing a plasma DNA sequencing library.

Description

A METHOD AND A KIT FOR QUICKLY CONSTRUCTING A PLASMA DNA SEQUENCING LIBRARY

Field of the Invention

The present invention is directed to a method and a kit for quickly constructing a plasma DNA sequencing library. More specifically， the present invention is directed to a method and a kit for quickly constructing a plasma DNA library for high throughput sequencing.

Background of the Invention

With the technological improvement， the traditional Sanger sequencing has been unable to fully meet the requirement of research. With regard to genome sequencing， there is a need for a sequencing technology with lower cost， higher throughput， and faster speed. Thus， a high throughput sequencing technology (also known as the second generation sequencing) emerges. The main principle of the high throughput sequencing technology is sequencing by synthesis， namely， determining DNA sequences by capturing newly synthesized end labelling. The available technologic platform mainly includes Roche/454 FLX， Illumina/Hiseq， Miseq， NextSeq and Life Technologies/SOLID system， PGM， Proton， and the like. Up to now， HiSeq 2000 can reach a sequencing throughput of 30X coverage in 6 human genomes per run， i.e.， about 600 G/run. With respect to sequencing time， Hiseq 2500 can reach a speed of reading a base per 8 minutes on average. Furthermore， with the maturity of the second generation sequencing technology， the investigation on its clinical application has developed quickly.

Plasma DNA， also known as circulating DNA， is an extracellular DNA in the blood and is tens to hundreds of nucleotides in length. It presents in the form of DNA-protein complex， or as free DNA fragment. Normally， plasma DNA is derived from DNA release of a small amount of senescent and dead cells. Under healthy condition， the generation and removal of the circulating DNA are in dynamic equilibrium and are maintained at a relatively steady low level. The circulating DNA can reflect the metabolic condition of cells in human body， thus is an important index for judging health. The change of quantity and quality of circulating DNA in peripheral blood is closely related to several diseases (including tumour， complex severe traumata， organ transplantation， pregnancy-related diseases， infectious diseases， organ failure， and the like) . As a non-invasive detection index， it is expected to be an important molecular marker for early diagnosing some diseases， monitoring the diseases and evaluating the therapeutic effects and prognosis of the diseases.

After the discovery of embryonic DNA in maternal blood， non-invasive direct diagnosis and detection of embryonic chromosomal abnormality has become an important research subject. In 2008， Professor Yuming LU and his team firstly examined the plasma DNA of a pregnant woman by a high throughput sequencing method， and detected the change of chromosome numbers of trace embryonic DNA in plasma by calculating the chromosome proportions (Chiu， Chan et al. 2008) . With gradual perfection and maturity of the detection technique， various countries are in an effort to convert the laboratory technique into clinical application. Additionally， it is found that the apoptosis products of cancer cells in a cancer patient (including fractured free cancer cell DNA) are released into blood， and then are delivered to the whole body via blood circulation system. Plasma DNA carries all the genetic information of the cancer cell genomes (Schwarzenbach， Hoon et al. 2011) . Recently， Professor Yuming LU distinguished cancer patients and healthy individuals by carrying out high throughput sequencing on the plasma DNA of cancer patients to calculate the methylation condition of the repeats (Chan， Jiang et al. 2013) .

The increase of sequencing efficiency and the popularization of mixing sequencing of multi-samples requires higher preparation efficiency of the samples， in particular the preparation of a large amount of clinical samples. However， the development of the method for preparing the clinical plasma samples nowadays fails to catch up with the increase of sequencing capability at all time. Therefore， the preparation efficiency and cost of the clinical plasma DNA samples in the second generation high throughput sequencing become the key of whether the high throughput sequencing can be popularized.

The preparation process of plasma DNA sample for the second generation high throughput sequencing substantively lies in inserting a DNA with a suitable sequencing length into a known sequencing vehicle， namely， adding linkers with known sequence to both ends of a DNA to be sequenced. Currently， the construction of a plasma DNA library mainly includes key steps such as extraction of a plasma DNA， terminal repair and 5’ terminal phosphorylation， then the addition of A on the end， connection of the linkers， PCR and the like (Fig. 1 ) ， in which a purification step is needed almost between every two steps. Such a method for constructing a plasma DNA sequencing library totally needs 6 main enzymes， 4 enzyme reaction systems， and 4 purification processes， thus result in higher cost and complex operation. Additionally， it requests for more strict condition， is easier to form aerosol pollution， and meantime requires higher operational capacity in molecular biology for the experimenter. Moreover， it is more difficult to achieve simultaneous processing of multiple samples. Furthermore， aerosol pollution caused during the PCR process is extremely easily to contaminate the sequencing sample， and the PCR process may also introduce a certain base preference， thereby causing asymmetry of sequencing results in coverage on the genome and finally resulting in inaccuracy in the results.

Summary of Invention

In view of the problems currently encountered in the process for constructing a plasma DNA sequencing library， the inventor found a new simpler， quicker， and evener method for constructing a plasma DNA sequencing library， which is applicable to multiple second generation sequencing platforms， including， but not limited to， Roche/454 FLX， Illumina/Hiseq， Miseq and Life Technologies/SOLID system， PGM， Proton and the like.

The invention is based on the following facts： the inventor has found that the plasma DNA can be sequenced after connected to a sequencing linker without PCR amplification； the amplification step in the current experimental process is not necessary； and the use of PCR polymerase is not necessary either. The inventor has also found that the three steps of end-filling， addition of A on the end and the connection of sequencing linkers in the current experimental process can be completed in one reaction system without purification step therebetween. Additionally， the end-filling process of the plasma DNA can be carried out without 5′end phosphorylation. The library to be sequenced can be obtained after the final purification. According to the present invention， a plasma DNA library can be well constructed and effective sequencing results can be obtained even when the amount of fractured plasma DNA within an organism is as low as 2 ng. Moreover， the present invention firstly applies PCR free technology to extremely low amount (less than 10 ng) of plasma DNA.

Therefore， in one aspect， the present invention provides a method for constructing a plasma DNA sequencing library， comprising the following steps：

1) Extracting a plasma DNA；

2) Optionally end-filling the plasma DNA to obtain an end-filled plasma DNA；

3) Adding A on the 3′end of the optionally end-fillied plasma DNA to obtain a3′end-added A plasma DNA；

4) Connecting the 3′end-added A plasma DNA with a sequencing linker to obtain a connected product； and

5) Purifying the connected product to obtain a purified product.

Thus， according to the present invention， the plasma DNA sequencing library can be obtained without the reaction step of PCR amplification of the purified product.

According to a preferred embodiment of the present invention， step 4) of connecting the 3′end-added A plasma DNA with a sequencing linker to obtain a connected product is carried out by one or two enzymes selected from the group consisting of T4 DNA ligase and T7 DNA ligase.

According to a preferred embodiment of the present invention， the sequencing linker is a double-stranded sequencing linker.

The double-stranded linker according to the present invention is obtained by annealing two single-stranded DNAs： one is a universal sequence， namely， this DNA has fixed sequences and has an identical application in different libraries， such as the universal adaptor shown hereinafter； the other one is a sequence with an index， namely， the sequence of this DNA has its specific bases (generally 3-8 specific bases) to distinguish between different linker sequences， wherein the sequences other than the specific bases are fixed， such as the index adaptor shown hereinafter.

Examples of linker sequences used in the present invention are as follows.

Universal Adapter

5′AATGATACGG CGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3′

Index adaptor

5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

The above linker sequences are merely examples of linker sequences of the present invention. According to the different sequencing platforms， such as Illumina， Ion Torrent， and Roche 454， those skilled in the art can connect different and suitable linker sequences to the plasma DNA library of the invention.

According to a preferred embodiment of the present invention， after extracting the plasma DNA and before connecting the plasma DNA with a sequencing linker， A is added on the end of the extracted plasma DNA， and the end-added A plasma DNA is purified. Preferably， a klenow ex-enzyme， a Taq enzyme， or a combination thereof is used during the addition of A on the end. Preferably， the addition of A on the end and the connection with sequencing linkers can be carried out in one reaction system， namely， the connection of sequencing linkers can be directly carried out after the addition of A on the end without purification. After the connection reaction， the plasma DNA is purified to obtain the plasma DNA library to be sequenced. Taq enzyme is used during the addition of A on the end.

According to a preferred embodiment of the present invention， after extracting the plasma DNA and before connecting the plasma DNA with a sequencing linker， the extracted plasma DNA is end-filled and A is added on the end. The end-filling and the addition of A on the end can be carried out in two reaction systems， namely， the end-filled plasma DNA is purified before the addition of A on the end. Alternatively and preferably， the end-filling and the addition of A on the end are carried out in one reaction system. After the end-filling and the addition of A on the end， the plasma DNA is purified. T4 DNA polymerase is used during the end-filling， and Taq enzyme is used during the addition of A on the end. More preferably， the end-filling and the addition of A on the end as well as connecting the plasma DNA with the sequencing linker are carried out in one reaction system. After the connection， the plasma DNA is purified to obtain the plasma DNA library to be sequenced. In the most prefer embodiment， the plasma DNA merely needs one reaction system， and the final sequencing library can be obtained after once purification. T4 DNA polymerase is used during the end-filling， and Taq enzyme is used during the addition of A on the end.

In another aspect， the present invention provides a kit for constructing a plasma DNA sequencing library， comprising：

-Reagents for optionally end-filling the plasma DNA， including dNTP， enzymes for the end-filling， and buffers for the end-filling；

-Reagents for adding A on the 3′end， including dATP， enzymes for adding A on the end， and buffers for adding A on the end；

-Reagents for connecting a sequencing linker， including sequencing linkers， ligases， and connecting buffer； and

-Reagents and devices for purifying the connected product.

Thus， according to the present invention， the kit does not comprise polymerase for PCR amplification.

According to an embodiment， the present invention provides a kit for constructing a plasma DNA sequencing library， an important feature of the kit is that the plasma DNA connected with the linker becomes a library to be sequenced after purification without the step of PCR amplification.

According to the kit of the invention， preferably， the ligase is one or two enzymes selected from the group consisting of T4 DNA ligase and T7 DNA ligase.

According to the kit of the invention， the sequencing linker is a double-stranded sequencing linker.

According to an embodiment， preferably， a klenow ex-enzyme， a Taq enzyme， or a combination thereof is used during the addition of A on the end.

In another embodiment， the kit of the present invention comprises： reagents for end-filling and adding A on the end of the extracted plasma DNA， including enzymes for the end-filling， dNTP， buffers for the end-filling， dATP， enzymes for adding A on the end， and buffers for adding A on the end； and reagents and devices for purifying the plasma DNA after the end-filling and the addition of A on the end. The end-filling and the addition of A on the end are carried out in one reaction system， whereinT4 DNA polymerase is used during the end-filling， and Taq enzyme is used during the addition A on the end.

In yet another embodiment， the kit of the present invention comprises： reagents for end-filling and adding A on the end of the extracted plasma DNA， including enzymes for the end-filling， dNTP， buffers for the end-filling， dATP， enzymes for adding A on the end， and buffers for adding A on the end； and reagents for connecting the plasma DNA with a sequencing linker， including sequencing linkers， ligases， and connecting buffers. The end-filling and the addition of A on the end as well as connecting the plasma DNA with the sequencing linker are carried out in one reaction system， wherein T4 DNA polymerase is used during the end-filling， and Taq enzyme is used during the addition A on the end.

According to an embodiment， the kit does not comprise a polymerase for PCR amplification， and does not comprise purification reagents and devices after the end repair and the addition of A on the 3′end.

According to an embodiment， reagents for purifying the connected product is sterile dH₂O or an elution buffer； devices for purifying the connected product is a purification column， a Qiagen column， purification magnetic beads or Beckman Ampure XP beads.

Based on the discovery that the plasma DNA connected with a linker can be sequenced directly， the present invention omits PCR reaction step from the existing method for constructing a plasma DNA sequencing library and no longer uses PCR polymerase. Additionally， based on this， the present invention replaces the klenow ex-enzyme by a conventional Taq enzyme for the addition of A on the end， so that various reaction systems become compatible： the end-filling， the addition of A on the end and the connection of sequencing linkers can be completed in one reaction system without purification step therebetween. Therefore the present invention greatly simplifies the process for constructing a plasma DNA sequencing library， reduces sample confusion and contamination caused by manual operation， and thus is more suitable for large-scale operation of plasma DNA library construction. Meanwhile， the present invention removes the contamination of environment as well as among samples due to PCR amplification， reduces the requirement for environment during the plasma DNA library construction， and removes the base preference， asymmetry coverage on the genome and the inaccuracy of the sequencing results due to PCR amplification. The present invention enables plasma sample library construction to be cheaper， more efficient and quicker， and also enables plasma DNA sequencing results to be more authentic and effective. Therefore， the present invention is more suitable for large-scale experimental operation and sequential analysis of the plasma DNA.

Brief Descrintion of Drawings

Fig. 1 shows the method for constructing the second generation high throughput plasma DNA sequencing library commonly used in the prior art.

Fig. 2 shows the method for constructing the second generation high throughput plasma DNA sequencing library according to one embodiment of the invention.

Fig. 3 shows the comparison of two different methods for constructing a library according to the prior art and one embodiment of the invention.

Fig. 4 shows the comparison of four different methods for constructing a library according to Examples 1-4 of the invention.

Embodiments

As mentioned above， the method for constructing a plasma DNA library applicable to the second generation high throughput sequencing platform used in the prior art mainly includes the following steps (Fig. 1) ： extracting a plasma DNA (step 101) → end-repairing (filling) the extracted plasma DNA， phosphorylating 5’ end， and purifying (step 102) → Adding A on the 3′end of the 5’ end-phosphorylated DNA fragment， and purifying (step 103) → connecting the 3′end-added A DNA with a sequencing linker (step 104) → purifying the connected product to remove the un-connected linkers (step 105) → carrying out PCR reaction with the purified product (step 106) → purifying the PCR product to obtain a plasma DNA sequencing library (step 107) . During the process， a purification step is needed almost between every two steps， and the plasma DNA is subject to PCR amplification finally. Thus， the method for constructing the second generation plasma DNA sequencing library totally needs 6 main enzymes， 4 enzyme reaction systems， 4 purification processes and 1PCR amplification， thus has a higher cost and complex operation. Additionally， it requests for more strict condition， is easier to form aerosol pollution， and meantime requires higher operational capacity in molecular biology for the experimenter. Moreover， it is more difficult to achieve simultaneous processing of multiple samples. Furthermore， aerosol pollution caused during the PCR process is extremely easily to contaminate the sequencing sample， and the PCR process may also introduce a certain base preference， thereby causing asymmetry of sequencing results in coverage on the genome and finally resulting in inaccuracy in the results.

After repeated experiments， the inventor has found that the plasma DNA can be sequenced after attached to a sequencing linker without PCR amplification. The amplification step in the current experimental process is not necessary. Thus， an important feature of the present invention for constructing a plasma DNA sequencing library is that the present invention does not comprise the step of PCR amplification.

Based on this， the inventor has also found that， according to the selection of the enzyme used in the addition of A on the end， the three steps of end-filling， addition of A on the end and the connection of sequencing linkers in the current experimental process do not need to be carried out in three reaction systems， but can be completed in one reaction system without purification step therebetween.

Fig. 2 shows the method for constructing the second generation high throughput plasma DNA sequencing library according to one embodiment of the invention， mainly includes the following steps (Fig. 2) ： extracting a plasma DNA (step 201) → end-filling and adding A on the end of the extracted plasma DNA (step 203) → connecting the 3′end-added A plasma DNA with a sequencing linker (step 204) → purifying the plasma DNA connected with a linker to obtain a plasma DNA sequencing library (step 207) . According to another embodiment， step 203 can be carried out in two reaction systems， namely， step 202 (end-repairing the extracted plasma DNA， and phosphorylating 5’ end) is carried out before step 203 (adding A on the 3′end of the 5’ end-phosphorylated DNA fragment) ； or a purification step is carried out between

steps

203 and 204.

Compared to the method for constructing a plasma DNA sequencing library disclosed in the prior art， the method of the present invention merely needs one reaction system and one step of purification without PCR process to obtain the final library to be sequenced. Therefore the present invention greatly simplifies the process for constructing a plasma DNA sequencing library， removes the base preference and asymmetry coverage on the genome due to the introduction of PCR steps. The present invention enables the plasma sample library construction to be cheaper， more efficient and quicker， and also enables the plasma DNA sequencing results to be more authentic and effective， thus is more suitable for large-scale use.

The present invention will be described in detail referring to the figures and examples. It should be noted that， one skilled in the art should know that the figures and examples of the present invention are provided only for the purpose of illustration， but do not constitute any limitation to the invention. Reconcilably， the examples of the application and the features of the examples can be combined with each other.

Example 1

The method for constructing a plasma DNA sequencing library of example 1 mainly includes the following steps (Fig. 4) ：

(1) Extracting a plasma DNA： this step can be carried out using any methods and reagents well known to one skilled in the art and suitable for extracting a plasma DNA.

(2) End-filling the plasma DNA， then cleaning and purifying the product： this step can be carried out using any methods and reagents well known to one skilled in the art and suitable for end-filling， cleaning and purifying. For example， T4 DNA polymerase and Klenow enzyme can be used as the enzyme for end-filling.

(3) Adding A on the end， then cleaning and purifying the product： this step can be carried out using any methods and reagents well known to one skilled in the art and suitable for adding A on the end， cleaning and purifying. For example， A base can be added on the double-stranded end of the product obtained from step (2) in the presence of klenow ex-enzyme (New England Biolabs) (it is an improved enzyme lacking 3’ -5’ exonuclease activity) .

(4) Connecting with a sequencing linker， then cleaning and purifying the product to obtain a plasma DNA library： this step can be carried out using any methods and reagents well known to one skilled in the art and suitable for connecting a linker， cleaning and purifying. For example， the end-added A product can be connected with a double-stranded sequencing linker in the presence of T4 DNA ligase， T7 DNA ligase or both. The sequencing linker is commercially available or can be obtained by synthesis.

Examples of linker sequences used in the present invention are as follows.

Universal Adapter

5′AATGATACGG CGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3′

Index adaptor

5′GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

Additionally， the following steps are required before the above library subject to high throughput sequencing：

(5) Quantifying the sequencing library using qPCR， and the concentration of the library should be greater than or equal to 20pM.

(6) Mixing the samples equally according to the arrangement of the channels and the difference of the tag numbers.

(7) Injecting the samples.

A specific example for sequencing the plasma DNA sample according to the method of Example 1 of the present invention is shown below.

Step 1： about 5 ng plasma DNA was extracted.

Step 2： a mixture for end-filling reaction as shown in Table 1 was prepared and placed in a 20℃ warm bath for 30min. The DNA sample was purified on a purification column and eluted by 32μl sterile dH₂O or elution buffer. The elution buffer used here was 10mM Tris-Cl， pH 8.0. However， the elution buffers suitable for the invention are not limited to this.

Table 1

Step 3： a reaction mixture for adding a poly-adenine tail on the 3’a s shown in Table 2 was prepared and placed in a 37℃ warm bath for 30min. The DNA sample was purified on a purification column and eluted by 37μl sterile dH₂O or elution buffer.

Table 2

Step 4： a reaction mixture for connecting the DNA fragment with a sequencing linker as shown in Table 3 was prepared and placed in a 20℃ warm bath for 15min， followed by a 65℃ warm bath for 10min， and was then maintained at 4℃. The connected plasma DNA sample was recovered by Beckman Ampure XP beads and eluted by 22μl sterile dH₂O or elution buffer.

Table 3

Step 5： Quantification and mix of the library： 1 μL library was sampled and quantified by qPCR， and it was qualified if the concentration of the library was greater than or equal to 20pM. The samples with different linkers in one channel were mixed equally according to the arrangement of the channels. 1μL mixture was sampled and quantified again by qPCR.

Step 6： Sequencing： the first end sequencing primer， tag primer and the second end sequencing primer were diluted into 100μM and sequenced according to the operation instruction of Illmina Hiseq2000. Alternatively， double-ended sequencing or single-ended sequencing can be carried out. The second end sequencing primer is not required when the single-ended sequencing is carried out.

Results： the sequencing results showed that the DNA sequencing library constructed in Example 1 of the present invention satisfied the design requirements of the second generation high throughput sequencing.

Example 2

The method for constructing a plasma DNA sequencing library according to Example 2 is substantively similar to that of Example 1， except that Example 2 omitted the step of end-filling. The extracted plasma DNA was directly added A and purified (see Fig. 4) .

A specific example for sequencing the plasma DNA sample according to the method of Example 2 of the invention is shown below.

Step 1： about 5 ng plasma DNA was extracted.

Step 2： a reaction mixture as shown in Table 4 was prepared and placed in a 37℃ warm bath for 30min for adding a poly-adenine tail on the 3’ end of the DNA fragment. The DNA sample was purified on a purification column and eluted by 37μl sterile dH₂O or elution buffer.

Table 4

Step 3： a reaction mixture for connecting the DNA fragment with a sequencing linker as shown in Table 5 was prepared and placed in a 20℃ warm bath for 15rmin， followed by a 65℃ warm bath for 10min， and was then maintained at 4℃. The connected plasma DNA sample was recovered by Beckman Ampure XP beads and eluted by 22μl sterile dH₂O or elution buffer.

Table 5

Step 4： Quantification and mix of the library： 1μL library was sampled and quantified by qPCR， and it was qualified if the concentration of the library was greater than or equal to 20pM. The samples with different linkers in one channel were mixed equally according to the arrangement of the channels. 1μL mixture was sampled and quantified again by qPCR.

Step 5： Sequencing： the first end sequencing primer， tag primer and the second end sequencing primer were diluted into 100μM and sequenced according to the operation instruction of Illmina Hiseq2000. Alternatively， double-ended sequencing or single-ended sequencing can be carried out. The second end sequencing primer is not required when the single-ended sequencing is carried out.

Results： the sequencing results showed that the DNA sequencing library constructed in Example 2 of the present invention satisfied the design requirements of the second generation high throughput sequencing.

Example 3

The method for constructing a plasma DNA sequencing library according to Example 3 is substantively similar to that of Example 1， except that in Example 3， the end-filling and the addition A on the end were carried out in one reaction system without cleaning and purification steps therebetween. The commonly used klenow ex-enzyme was replaced by a conventional Taq enzyme for the addition of A on the end， so that the two reaction systems became compatible (Fig. 4) .

A specific example for sequencing the plasma DNA sample according to the method of Example 3 of the invention is shown below.

Step 1： about 5 ng plasma DNA was extracted.

Step 2： a reaction mixture as shown in Table 6 was prepared and placed in a 37℃ warm bath for 30min (for end-filling) and in a 72℃ warm bath for 20min (for adding A on the end) ， so that the end-filling and the addition of A on the end were carried out in one reaction system. The DNA sample was purified on a purification column and eluted by 37μl sterile dH₂O or elution buffer. The temperature and time for the end-filling and the addition of A on the end can vary according to the compositions of different reaction mixtures suitable for different design requirements.

Table 6

Step 3： a reaction mixture for connecting the DNA fragment with a sequencing linker as shown in Table 7 was prepared and placed in a 20℃ warm bath for 15min， followed by a 65℃ warm bath for 10min， and was then maintained at 4℃. The connected plasma DNA sample was recoverd by Beckman Ampure XP beads and eluted by 22μl sterile dH2O or elution buffer.

Table 7

Step 4： Quantification and mix of the library： 1 μL library was sampled and quantified by qPCR， and it was qualified if the concentration of the library was greater than or equal to 20pM. The samples with different linkers in one channel were mixed equally according to the arrangement of the channels. 1 μL mixture was sampled and quantified again by qPCR.

Results： the sequencing results showed that the DNA sequencing library constructed in Example 3 of the present invention satisfied the design requirements of the second generation high throughput sequencing.

Example 4

The method for constructing a plasma DNA sequencing library according to Example 1 was substantively similar to that of Example 1， except that in Example 4， the end-filling， the addition of A on the end and the connection with a sequencing linker were carried out in one reaction system without cleaning and purification steps between the end-filling and the addition A on the end， or between the addition of A on the end and the connection with a sequencing linker. The commonly used klenow ex-enzyme was replaced by a conventional Taq enzyme for the addition of A on the end， so that the three reaction systems became compatible (see Fig. 4) .

A specific example for sequencing the plasma DNA sample according to the method of Example 4 of the invention is shown below.

Step 1： about 5 ng plasma DNA was extracted.

Step 2： a reaction mixture as shown in Table 8 was prepared and placed in a 37℃ warm bath for 30min (for end-filling) and in a 72℃ warm bath for 20min (for adding A on the end) ， so that the end-filling and the addition of A on the end were completed without a purification step.

Table 8

Step 3： an additional reaction reagent was added into the above reaction solution. A reaction mixture for connecting the DNA fragment with a sequencing linker as shown in Table 9 was prepared and placed in a 20℃ warm bath for 15min， followed by a 65℃warm bath for 10min， and was then maintained at 4℃. The connected plasma DNA sample was recovered by Beckman Ampure XP beads and eluted by 22μl sterile dH₂O or elution buffer.

Table 9

Step 4： Quantification and mix of the library： 1μL library was sampled and quantified by qPCR， and was qualified if the concentration of the library is greater than or equal to 20pM. The samples with different linkers in one channel were mixed equally according to the arrangement of the channels. 1 μL mixture was sampled and quantified again by qPCR.

Results： the sequencing results showed that the DNA sequencing library constructed in Example 4 of the present invention satisfied the design requirements of the second generation high throughput sequencing.

Moreover， the invention also provides a kit for quickly constructing a plasma DNA sequencing library according to Examples 1-4. According to different examples， the kit can include enzymes for filling and repairing the ends of plasma DNA； enzymes for adding an adenine on the 3’ end of the DNA， including T4 DNA polymerase， Klenow enzyme， DNA polymerase， Taq enzyme and klenow ex-enzyme (lacking 3’ -5’ exonuclease activity) ； enzymes for connecting linkers， including T4 DNA ligase， T7 DNA ligase and a combination thereof； and buffers required by the various enzymes； as well as dATP for end-filling and the addition of A on the end， double-stranded sequencing linkers for connecting a linker， and the like.

Since the inventor found that the construction of a plasma DNA sequencing library does not require a reaction step of PCR amplification， an important feature of the kit of the present invention is that it does not comprise a polymerase for PCR amplification.

Moreover， by adding A on the end using Taq enzyme， the invention achieves that the end-filling and the addition of A on the end are carried out in one reaction system， or that the end-filling and the addition of A on the end as well as the connection with sequencing linkers are carried out in one reaction system. Thus， the kit of the present invention may not include reagents and devices for purifying the end-filled products and/or may not include reagents and devices for purifying the end-added A products， thereby reducing the cost and simplifying the process for constructing a library.

Under necessary conditions， the kit of the present invention may further comprise reagents and devices for purifying the end-filled products； reagents and devices for purifying the end-added A products； and reagents and devices for purifying the products connected with a sequencing linker. The reagents for purification may include sterile dH₂O or an elution buffer； devices for purification may include a purification column， a Qiagen column， purification magnetic beads or Beckman Ampure XP beads. The reagents and devices for purification are not limited to those specifically listed here. Various reagents and devices commonly used in the art can be used in the present invention according to the judgement of those skilled in the art.

It should be noted that the above examples are merely the preferred examples of the invention and are not intended to limit the invention. Those skilled in the art can modify and change the invention appropriately. For example， the steps of the addition of A on the ends and the connection with the sequencing linkers can be carried out via two reactions and in one system， namely， the extracted plasma DNA can be added A on the end and connected with the linkers without purification step therebetween. Additionally， although the enzymes for connecting sequencing linkers shown herein are T4 DNA ligase and/or T7 DNA ligase， T7 DNA ligase alone or in combination with T4 DNA ligase can be used. The reaction buffers and the like used during the construction of a plasma DNA sequencing library can be adjusted and changed according to specific requirements. Those skilled in the art will understand that any modification， equivalent replacement and improvement within the spirit and principle of the invention should be contained in the protection scope of the invention.

References：

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Claims

A method for constructing a plasma DNA sequencing library， comprising the following steps：

1) Extracting a plasma DNA；

2) Optionally end-filling the plasma DNA to obtain an end-filled plasma DNA；

3) Adding A on the 3′ end of the optionally end-filled plasma DNA to obtain a 3′ end-added A plasma DNA；

4) Connecting the 3′ end-added A plasma DNA with a sequencing linker to obtain a connected product； and

5) Purifying the connected product to obtain a purified product.
The method according to claim 1， characterized in that the step 4) is carried out by one or two enzymes selected from the group consisting of T4 DNA ligase and T7 DNA ligase.
The method according to claim 1， characterized in that the sequencing linker is a double-stranded sequencing linker.
The method according to claim 1， characterized in that a klenow ex-enzyme， a Taq enzyme， or a combination thereof is used during the addition of A on the end.
The method according to claim 1， characterized in that a purification step is carried out between the steps 2) and 3) ； or the steps 2) and 3) are carried out in one reaction system without a purification step therebetween.
The method according to claim 5， characterized in that T4 DNA polymerase is used during the step 2) ， and Taq enzyme is used during the step 3) .
The method according to claim 1， characterized in that a purification step is carried out between the steps 3) and 4) ； or the steps 2) ， 3) and 4) are carried out in one reaction system without a purification step thereamong.
The method according to claim 7， characterized in that T4 DNA polymerase is used during the step 2) ， Taq enzyme is used during the step 3) ， and T4 DNA ligase and T7 DNA ligase are used during the step 4) .
A kit for constructing a plasma DNA sequencing library， comprising：

-Reagents for optionally end-filling the plasma DNA， including dNTP， enzymes for the end-filling， and buffers for the end-filling；

-Reagents for adding A on the 3′ end， including dATP， enzymes for adding A on the end， and buffers for adding A on the end；

-Reagents for connecting with a sequencing linker， including sequencing linkers， ligases， and connecting buffer；

-Reagents and devices for purifying the connected product.
The kit according to claim 9， characterized in that the ligase is selected from the group consisting of T4 DNA ligase and T7 DNA ligase.
The kit according to claim 9， characterized in that the sequencing linker is a double-stranded sequencing linker.
The kit according to claim 9， characterized in that the enzyme used during the addition of A on the ends is a klenow ex-enzyme， a Taq enzyme， or a combination thereof.
The kit according to claim 9， characterized in that the enzyme used during the end-filling is T4 DNA polymerase.
The kit according to claim 12 or 13， characterized in that the enzyme used during the end-filling is T4 DNA polymerase； and the enzyme used during the addition of A on the end is Taq enzyme.
The kit according to claim 9， characterized in that the enzyme used during the end-filling is T4 DNA polymerase； the enzyme used during the addition of A on the end is Taq enzyme； the enzyme for connection is T4 DNA ligase and T7 DNA ligase； and the linker for connection is a double-stranded sequencing linker.
The kit according to claim 9， characterized in that the reagents for purifying the connected product is sterile dH₂O or an elution buffer； the devices for purifying the connected product is a purification column， a Qiagen column， purification magnetic beads or Beckman Ampure XP beads.