Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction

Abstract

The invention generally relates to methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction. In certain embodiments, methods of the invention involve obtaining a template nucleic acid, incorporating a pair of sequence identifiers into the template, and sequencing the template.

1 - 46 . (canceled) 
     
     
         47 . A method for analyzing a target polynucleotide sequence, the method comprising:
 i) attaching at least one unique pair of identifier sequences to the target polynucleotide sequence from a sample,   wherein at least a first identifier sequence is incorporated into a 5′ portion of the target polynucleotide sequence and wherein at least a second identifier sequence is incorporated into a 3′ portion of the target polynucleotide sequence, and   wherein the first identifier sequence and second identifier sequence each have four or more nucleotides and are different;   ii) pooling the target polynucleotide sequence with other samples to form a pooled nucleic acid library in which each sample is tagged with a unique sample-specific identifier sequence;   iii) hybridizing the pooled nucleic acid library to primers attached to discrete locations on a flow cell;   iv) amplifying the pooled nucleic acid library to form clusters of amplicons attached to the surface of the flow cell, wherein the plurality of amplicons comprise the first identifier sequence, the target polynucleotide sequence and the second identifier sequence;   v) sequencing each amplicon of the plurality of amplicons to form a sequence data set;   vi) analyzing the sequence data set and determining the first identifier sequence and the second identifier sequence of each amplicon; and   vii) identifying a chimeric molecule in the plurality of amplicons when only one identifier is detected or an incorrect pair of identifier sequences are detected.   
     
     
         48 . The method of  claim 47 , further comprising:
 viii) discarding the sequence data of the chimeric molecule from the sequence data set.   
     
     
         49 . The method of  claim 47 , wherein the identifier sequences are nucleic acid sequence tags. 
     
     
         50 . The method of  claim 49 , wherein the identifier sequences are barcode sequences. 
     
     
         51 . The method of  claim 50 , wherein the barcode sequences are from 4 to 20 nucleotides in length. 
     
     
         52 . The method of  claim 51 , wherein the barcode sequences are designed to have no homopolymer regions.