Method and kit for constructing plasma dna sequencing library

Abstract

The disclosure relates a method and a kit for constructing a plasma Deoxyribonucleic acid (DNA) sequencing library. The method provided by the disclosure includes: extracting a plasma DNA; making the plasma DNA ligate to a sequencing linker, and purifying a ligation product; performing Polymerase Chain Reaction (PCR) amplification for the purified ligation product, purifying the PCR amplification product, and obtaining the plasma DNA sequencing library, wherein, the method does not include the step of performing 5′-terminus phosphorylation for the plasma DNA. The kit provided by the disclosure includes: a reagent which ligates a plasma DNA to a sequencing linker, including the sequencing linker, a ligase and a ligation buffer; and reagents and instruments for purifying the ligation product; a reagent which performs PCR amplification for a purified ligation product, and reagents and instruments for purifying the PCR amplification product; wherein, the kit does not include the reagent which performs 5′-terminus phosphorylation for the plasma DNA. The disclosure simplifies the construction flow of a plasma DNA sequencing library, simplifies the experimental procedures, and makes the construction of the plasma sample library have lower cost, higher efficiency and faster speed, and is convenient for large-scale application.

1 . A method for constructing a plasma Deoxyribonucleic acid (DNA) sequencing library, comprising:
 extracting a plasma DNA;   ligating the plasma DNA with a sequencing linker, and purifying a ligation product;   performing Polymerase Chain Reaction (PCR) amplification for a purified ligation product, purifying a PCR amplification product, and obtaining the plasma DNA sequencing library;   wherein, the method does not comprise the step of performing 5′-terminus phosphorylation for the plasma DNA.   
     
     
         2 . The method according to  claim 1 , wherein, ligating the plasma DNA with the sequencing linker is performed by adopting one or more ligases selected from the group consisting of: a T4 DNA ligase, a T4 Ribonucleic Acid (RNA) ligase and a T7 DNA ligase. 
     
     
         3 . The method according to  claim 1 , wherein the sequencing linker is a single-strand sequencing linker. 
     
     
         4 . The method according to  claim 1 , wherein the sequencing linker is a double-strand sequencing linker. 
     
     
         5 . The method according to  claim 4 , wherein, after extracting the plasma DNA, and before ligating the plasma DNA with the sequencing linker, performing dA-overhang for the extracted plasma DNA, and purifying a plasma DNA with dA-overhang. 
     
     
         6 . The method according to  claim 5 , whereina the dA-overhang adopts klenow ex-enzyme, or Taq enzyme, or a combination of klenow ex-enzyme and Taq enzyme. 
     
     
         7 . The method according to  claim 4 , wherein, after extracting the plasma DNA, and before ligating the plasma DNA with the sequencing linker, the method further comprises performing end-filling and dA-overhang for the extracted plasma DNA. 
     
     
         8 . The method according to  claim 7 , wherein the method comprises performing the end-filling and dA-overhang in one reaction system, and then purifying an end-filled and dA-overhanged plasma DNA. 
     
     
         9 . The method according to  claim 8 , wherein T4 DNA polymerase is adopted for the end-filling, and Taq enzyme is adopted for the dA-overhang. 
     
     
         10 . The method according to  claim 7 , wherein the method comprises performing the end-filling, the dA-overhang, and the ligation of the plasma DNA with the sequencing linker in one reaction system. 
     
     
         11 . The method according to  claim 10 , wherein T4 DNA polymerase is adopted for the end-filling, and Taq enzyme is adopted for the dA-overhang. 
     
     
         12 . A kit for constructing a plasma DNA sequencing library, comprising:
 a reagent which ligates a plasma DNA to a sequencing linker, the reagent comprising the sequencing linker, a ligase and a ligation buffer;   reagents and instruments for purifying a ligation product;   a reagent which performs PCR amplification for a purified ligation product and reagents and instruments for purifying a PCR amplification product;   wherein, the kit does not comprise a reagent which performs 5′-terminus phosphorylation for the plasma DNA.   
     
     
         13 . The kit according to  claim 12 , wherein, the ligase is selected from the group consisting of: T4 DNA ligase, T4 RNA ligase and T7 DNA ligase. 
     
     
         14 . The kit according to  claim 12 , wherein the sequencing linker is a single-strand sequencing linker. 
     
     
         15 . The kit according to  claim 12 , wherein the sequencing linker is a double-strand sequencing linker. 
     
     
         16 . The kit according to  claim 15 , wherein the kit further comprises:
 a reagent which performs dA-overhang for a extracted plasma DNA, the reagent comprising Deoxyadenosine Triphosphate (dATP), an enzyme for dA-overhang, a buffer for dA-overhang; and   reagents and instruments for purifying the plasma DNA with dA-overhang.   
     
     
         17 . The kit according to  claim 16 , wherein the enzyme for dA-overhang is klenow ex-enzyme, or Taq enzyme, or a combination of klenow ex-enzyme and Taq enzyme. 
     
     
         18 . The kit according to  claim 15 , further comprising:
 a reagent which performs end-filling and dA-overhang for the extracted plasma DNA, the reagent comprising an enzyme for end-filling, Diethyl-Nitrophenyl Thiophosphate (dNTP), a buffer for end-filling, Deoxyadenosine Triphosphate (dATP), an enzyme for dA-overhang, and a buffer for dA-overhang; and   reagents and instruments for purifying an end-filled and dA-overhanged plasma DNA.   
     
     
         19 . The kit according to  claim 15 , wherein, the kit further comprising:
 a reagent which performs end-filling and dA-overhang for the extracted plasma DNA in one reaction system, the reagent comprising an enzyme for end-filling, dNTP, a buffer for end-filling, dATP, an enzyme for dA-overhang, and a buffer for dA-overhang; and   the reagents and instruments respectively for purifying an end-filled and dA-overhanged plasma DNA.   
     
     
         20 . The kit according to  claim 19 , wherein, the enzyme for end-filling is T4 DNA polymerase; and the enzyme for dA-overhang is Taq enzyme. 
     
     
         21 . The kit according to  claim 15 , the kit further comprising:
 a reagent which performs end-filling and dA-overhang for the extracted plasma DNA, the reagent comprising an enzyme for end-filling, dNTP, a buffer for end-filling, dATP, an enzyme for dA-overhang, and a buffer for dA-overhang;   wherein the end-filling and dA-overhang, and the ligation between the plasma DNA and the sequencing linker, are performed in one reaction system.   
     
     
         22 . The kit according to  claim 21 , wherein, the enzyme for end-filling is T4 DNA polymerase; and the enzyme for dA-overhang is Taq enzyme.