High multiplex pcr with molecular barcoding

Abstract

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a molecular barcode and a target-specific sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence. Such methods avoid barcode resampling and suppress primer dimers in traditional high multiplex PCR.

1 . A kit comprising:
 (1) a plurality of barcode primers (BC primers), wherein
 (i) each barcode primer comprises, from 5′ to 3′, a 1 st  universal primer sequence (US1), a molecular tag sequence (MT), and a 1 st  target-specific sequence (TS1), 
 (ii) a plurality of barcode primers comprise at least 20 different barcode primers, and 
 (iii) among the plurality of barcode primers, the 1 st  universal primer sequence (US1) are the same, the molecular tag sequences (MT) are different, and the 1 st  target-specific sequence (TS1) are different; and 
   (2) a plurality of limited amplification primers (LA primers), wherein
 (i) each limited amplification primer comprises, from 5′ to 3′, a 2 nd  universal primer sequence (US2) and a 2 nd  target-specific sequence (TS2), and 
 (ii) among the plurality of limited amplification primers, the 2 nd  universal primer sequences (US2) are the same, but the 2 nd  target-specific sequence (TS2) are different. 
   
     
     
         2 . The kit of  claim 1 , wherein the plurality of barcode primers (BC primers) comprises at least 500 different barcode primers, and the plurality of limited amplification primers (LA primers) comprises at least 500 different limited amplification primers. 
     
     
         3 . The kit of  claim 1 , wherein the number of different barcode primers (BC primers) is the same as the number of different limited amplification primers (LA primers). 
     
     
         4 . The kit of  claim 1 , wherein the number of different barcode primers (BC primers) is different from the number of different limited amplification primers (LA primers). 
     
     
         5 . The kit of  claim 1 , wherein each of the 1 st  target-specific sequences of the plurality of barcode primers (BC primers) and the 2 nd  target-specific sequences of the plurality of limited amplification primers (LA primers) does not contain more than 10 nucleotides that are complementary to the 1 st  target-specific sequences of any other barcode primers or to the 2 nd  target-specific sequences of any other limited amplification primers. 
     
     
         6 . The kit of  claim 1 , wherein the molecular tag sequences (MT) in the plurality of barcode primers are completely or semi-defined. 
     
     
         7 . The kit of  claim 1 , wherein the molecular tag sequences (MT) in the plurality of barcode primers are completely random. 
     
     
         8 . The method of  claim 1 , wherein the molecular tag sequences (MT) in the plurality of barcode primers are 5 to 15 nucleotides in length. 
     
     
         9 . The kit of  claim 1 , further comprising: a primer comprising the 1 st  universal primer sequence (US1). 
     
     
         10 . The kit of  claim 1 , further comprising a pair of universal primers (Uni-primers), wherein one of the universal primers comprises at its 3′-terminus the 1 st  universal primer sequence (US1), and the other of the universal primers comprises at its 3′-terminus the 2 nd  universal primer sequence (US2). 
     
     
         11 . The kit of  claim 10 , wherein each of the universal primers comprises an adapter sequence. 
     
     
         12 . The kit of  claim 10 , wherein either one or both of the universal primers comprise an index sequence (IDX) located 5′ to the 1 st  universal primer sequence (US1) or the 2 nd  universal primer sequence (US2). 
     
     
         13 . The kit of  claim 1 , further comprising a 1 st  DNA polymerase. 
     
     
         14 . The kit of  claim 13 , wherein the 1 st  DNA polymerase does not have strand displacement activity, flap endonuclease or 5′43′ exonuclease activity. 
     
     
         15 . The kit of  claim 1 , further comprising a 2 nd  DNA polymerase. 
     
     
         16 . The kit of  claim 1 , further comprising dNTPs and one or more PCR reaction buffers.