Measurement of nucleic acid variants using highly-multiplexed error-suppressed deep sequencing

Abstract

Methods and compositions are disclosed for measuring low-abundance DNA variants from a complex mixture of DNA molecules. Embodiments of the methods allow for extremely sensitive detection and can distinguish true variants from sequencer misreads and PCR misincorporations.

1 .- 15 . (canceled) 
     
     
         16 . A method of identifying a DNA sequence error produced by a sequencing instrument, the method comprising:
 a) generating a plurality of clonal DNA clusters, wherein a clonal DNA cluster comprises a plurality of top strand DNA copies and a plurality of bottom strand DNA copies, wherein all copies are derived from a single template DNA molecule;   b) using the sequencing instrument to read a sequence from only the top strand DNA copies or only the bottom strand DNA copies within the clonal DNA cluster, thereby producing a first clonal sequence read;   c) using the sequencing instrument to separately read, within the same clonal DNA cluster, a sequence from DNA strands that are in the opposite orientation to those used to produce the first clonal sequence read, thereby producing a second clonal sequence read;   d) aligning the first clonal sequence read and the second clonal sequence read based on sequence complementarity in a region of expected sequence overlap; and   e) identifying the DNA sequence error at a position that lacks perfect sequence complementarity in the region of expected sequence overlap between the first clonal sequence read and the second clonal sequence read.   
     
     
         17 . The method of  claim 16 , wherein the first clonal sequence read and the second clonal sequence read overlap completely. 
     
     
         18 . The method of  claim 16 , wherein the first clonal sequence read and the second clonal sequence read overlap partially. 
     
     
         19 . The method of  claim 16 , wherein the lack of perfect sequence complementarity can occur due to mismatches at a plurality of nucleotide positions, a mismatch at a single nucleotide position, a deletion, or an insertion. 
     
     
         20 . The method of  claim 16 , wherein improvements in sensitivity and accuracy of quantification of low-abundance sequence variants are enabled by identifying the DNA sequence errors. 
     
     
         21 . The method of  claim 20 , wherein the low-abundance sequence variants comprise mutant tumor-derived DNA fragments extracted from blood. 
     
     
         22 . A method of synthesizing modular oligonucleotide primer mixes, the method comprising:
 a) synthesizing a plurality of 3′ oligonucleotide segments comprising a plurality of target-specific primer sequences, wherein each target-specific primer sequence is synthesized in a separate synthesis column on solid supports;   b) pausing the synthesis;   c) pooling and thoroughly mixing all solid supports from all synthesis columns containing the partially-synthesized 3′ oligonucleotide segments;   d) dispensing the pooled mixture of partially-synthesized 3′ oligonucleotide segments into a plurality of new synthesis columns;   e) resuming synthesis to add a 5′ oligonucleotide segment comprising a unique sample-specific barcode to each new synthesis column; and   f) cleaving and deprotecting the oligonucleotides from the solid supports in each column, yielding a plurality of modular oligonucleotide mixes, wherein each mix contains a unique, sample-specific barcode and a plurality of target-specific primer sequences.   
     
     
         23 . The method of  claim 22 , further comprising incorporation of a molecular lineage tag in the 5′ oligonucleotide segment. 
     
     
         24 . The method of  claim 22 , wherein the modular oligonucleotide primer mixes enable early assignment of sample-specific barcodes to a plurality of target sequences from a plurality of samples. 
     
     
         25 . The method of  claim 24 , wherein the target sequences comprise DNA. 
     
     
         26 . The method of  claim 24 , wherein the target sequences comprise RNA. 
     
     
         27 . The method of  claim 24 , wherein the plurality of samples comprise clinical specimens. 
     
     
         28 . The method of  claim 27 , wherein the clinical specimens are from different clinical patients. 
     
     
         29 . The method of  claim 27 , wherein the clinical specimens are from different clinical time points.