Methods and kits for nucleic acid sample preparation for sequencing

Abstract

The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.

1 . A method for DNA library construction, comprising:
 modifying nucleic acid fragments; and   
       ligating nucleic acid adaptors to the modified nucleic acid fragments; 
       wherein one or more of the reactions are rehydrated from a lyophilized form. 
     
     
         2 . The method of  claim 1 , comprising:
 modifying the DNA fragments with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;   contacting the end repaired DNA fragments with (3′→5′ exo-) polymerase in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;   ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising a nucleic acid ligase;   wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form.   
     
     
         3 . The method of  claim 2 , comprising:
 modifying DNA fragments with T4 DNA polymerase and T4 polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;   contacting the end repaired DNA fragments with Klenow (3′→5′ exo-) in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;   ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising T4 DNA ligase,   wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form.   
     
     
         4 . The method of  claim 2 , wherein at least the first reaction mixture is rehydrated from a lyophilized form. 
     
     
         5 . The method of  claim 4 , wherein the lyophilized form is one or more lyophilized pellets. 
     
     
         6 . The method of  claim 4 , wherein the lyophilized form comprises T4 DNA polymerase, T4 DNA polynucleotide kinase, deoxynucleotide triphosphates, a source of magnesium, and a buffer. 
     
     
         7 . The method of  claim 6 , wherein the rehydrated reaction mixture is from about 25 to about 150 μL volume. 
     
     
         8 . The method of  claim 7 , wherein the rehydrated reaction mixture is about 50 μl or about 100 μl in volume. 
     
     
         9 . The method of  claim 5 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         10 . The method of  claim 9 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         11 . The method of  claim 4 , wherein the DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture. 
     
     
         12 . The method of  claim 2 , wherein at least the second reaction mixture is rehydrated from a lyophilized form. 
     
     
         13 . The method of  claim 12 , wherein the lyophilized form is one or more lyophilized pellets. 
     
     
         14 . The method of  claim 12 , wherein the lyophilized form comprises Klenow (3′ →5′ exo-), a source of magnesium, a source of sodium, dATP, and a buffer. 
     
     
         15 . The method of  claim 14 , wherein the rehydrated reaction mixture is from about 25 to about 200 μl volume. 
     
     
         16 . The method of  claim 15 , wherein the rehydrated reaction mixture is about 50 or about 100 μl in volume. 
     
     
         17 . The method of  claim 13 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         18 . The method of  claim 17 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         19 . The method of  claim 12 , wherein the end repaired DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture. 
     
     
         20 . The method of  claim 2 , wherein at least the third reaction mixture is rehydrated from lyophilized form. 
     
     
         21 . The method of  claim 20 , wherein the lyophilized form is one or more lyophilized pellets. 
     
     
         22 . The method of  claim 20 , wherein the lyophilized form comprises T4 DNA ligase, a source of magnesium, ATP, and a buffer. 
     
     
         23 . The method of  claim 22 , wherein the rehydrated reaction mixture is from about 25 to about 200 μl volume. 
     
     
         24 . The method of  claim 23 , wherein the rehydrated reaction mixture is from about 50 to about 100 μl in volume. 
     
     
         25 . The method of  claim 21 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         26 . The method of  claim 25 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         27 . The method of  claims 20 , wherein the A-tailed DNA in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture. 
     
     
         28 . The method of  claim 2 , wherein at least two of the first reaction mixture, second reaction mixture, and third reaction mixture are rehydrated from lyophilized form. 
     
     
         29 . The method of  claim 28 , wherein each of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from lyophilized form. 
     
     
         30 . A kit for DNA library construction and for performing the method of  claim 1 , comprising:
 a reaction tube containing first reaction components for end repair of DNA fragments to be added;   a reaction tube containing second reaction components for A-tailing of end-repaired DNA fragments;   a reaction tube contain third reaction components for ligating DNA adaptors to A-tailed DNA;   wherein one, two, or each of the first reaction components, the second reaction components, and the third reaction components are in lyophilized form.