Methods for sample preparation

Abstract

The disclosure provides for single amplification and double amplification methods for preparing nucleic acid samples for sequencing.

What is claimed is: 
     
         1 . A method for generating an adaptor-ligated library of double-stranded nucleic acid molecules comprising, in a single reaction vessel or reaction mixture:
 (a) generating, from a plurality of single-stranded polynucleotides, a plurality of double-stranded DNA molecules by:
 i. contacting said plurality of polynucleotides with a pool of primers comprising a cleavable 5′ end; and 
 ii. extending said primers to generate first extension products hybridized to said plurality of polynucleotides thereby forming double-stranded duplexes, wherein said first extension products are formed from single template molecules; 
   (b) cleaving the cleavable 5′ end with a cleaving agent, thereby generating double-stranded DNA molecules, wherein said double-stranded DNA molecules have a blunt end and an overhang end; and   (c) ligating one or more double-stranded adapters, wherein a first set of adapters comprises a 3′ overhang complementary to the overhang end of the double-stranded DNA molecules and a second set of adapters does not comprise the 3′ overhang, to the double-stranded DNA molecules, thereby forming the adapter-ligated library using only a single round of extension; and, optionally,   (d) sequencing the adapter-ligated library,   
       wherein the plurality of single-stranded polynucleotides is present at a quantity less than about 5 nanograms. 
     
     
         2 . The method of  claim 1 , wherein the cleavable 5′ end is selected from the group consisting of: rA, rC, rG, rU, or dU. 
     
     
         3 . The method of  claim 1 , wherein the ligating occurs with an efficiency greater than 90%. 
     
     
         4 .- 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein the pool of primers comprises random primers. 
     
     
         14 . The method of  claim 1 , wherein the pool of primers comprises target-specific primers. 
     
     
         15 . The method of  claim 14 , wherein the target-specific primers comprise barcode sequence upstream of the target-specific sequence. 
     
     
         16 .- 30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein said cleaving agent is a uracil-DNA glycolyase. 
     
     
         32 . The method of  claim 1 , wherein said cleaving agent is an RNaseH. 
     
     
         33 . The method of  claim 1 , wherein said extending further comprises incorporating one or more cleavable dNTPs, thereby generating an extended strand comprising cleavable dNTPs. 
     
     
         34 . The method of  claim 33 , wherein said extended strand is cleaved and said sequencing does not comprise sequencing said extended strand. 
     
     
         35 . The method of  claim 34 , wherein sequencing reads from said sequencing do not comprise errors due to extension or PCR performed prior to said sequencing.