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 - 30 . (canceled) 
     
     
         31 . A method for nucleic acid library construction, the method comprising:
 end repairing nucleic acid fragments in a first reaction mixture;   A-tailing the end-repaired nucleic acid fragments in a second reaction mixture; and   ligating nucleic acid adaptors to the A-tailed nucleic acid fragments in a third reaction mixture,   wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments.   
     
     
         32 . The method of  claim 31 , wherein at least the first reaction mixture is rehydrated from a lyophilized form. 
     
     
         33 . The method of  claim 32 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         34 . The method of  claim 33 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         35 . The method of  claim 31 , wherein the first reaction mixture comprises a polymerase and a polynucleotide kinase. 
     
     
         36 . The method of  claim 35 , wherein the polymerase is a T4 DNA polymerase, and wherein the polynucleotide kinase is a T4 polynucleotide kinase. 
     
     
         37 . The method of  claim 35 , wherein the first reaction mixture further comprises deoxynucleotide triphosphates, a source of magnesium, and a buffer. 
     
     
         38 . The method of  claim 32 , wherein the nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture. 
     
     
         39 . The method of  claim 31 , wherein at least the second reaction mixture is rehydrated from a lyophilized form. 
     
     
         40 . The method of  claim 39 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         41 . The method of  claim 40 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         42 . The method of  claim 31 , wherein the second reaction mixture comprises a (3′→5′ exo-) polymerase. 
     
     
         43 . The method of  claim 42 , wherein the (3′→′ exo-) polymerase is a Klenow exo-). 
     
     
         44 . The method of  claim 42 , wherein the second reaction mixture further comprises a source of magnesium, a source of sodium, dATP, and a buffer. 
     
     
         45 . The method of  claim 39 , wherein the end-repaired nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture. 
     
     
         46 . The method of  claim 31 , wherein at least the third reaction mixture is rehydrated from a lyophilized form. 
     
     
         47 . The method of  claim 46 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm. 
     
     
         48 . The method of  claim 47 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume. 
     
     
         49 . The method of  claim 31 , wherein the third reaction mixture comprises a nucleic acid ligase. 
     
     
         50 . The method of  claim 49 , wherein the nucleic acid ligase is a T4 DNA ligase. 
     
     
         51 . The method of  claim 49 , wherein the third reaction mixture further comprises a source of magnesium, ATP, and a buffer. 
     
     
         52 . The method of  claim 46 , wherein the A-tailed nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture. 
     
     
         53 . The method of  claim 31 , wherein at least two of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form. 
     
     
         54 . The method of  claim 31 , wherein each of the first reaction mixture, the second reaction mixture, and the third reaction mixture is rehydrated from a lyophilized form. 
     
     
         55 . The method of  claim 31 , wherein the nucleic acid fragments comprise DNA fragments. 
     
     
         56 . A method for nucleic acid library construction, the method comprising:
 end repairing nucleic acid fragments in a first reaction mixture, wherein the first reaction mixture comprises a polymerase and a polynucleotide kinase;   A-tailing the end-repaired nucleic acid fragments in a second reaction mixture, wherein the second reaction mixture comprises a (3′→5′ exo-) polymerase; and   ligating nucleic acid adaptors to the A-tailed nucleic acid fragments in a third reaction mixture, wherein the third reaction mixture comprises 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 in a reaction volume comprising the nucleic acid fragments.