Detection of rare sequence variants, methods and compositions therefor

Abstract

The present disclosure encompasses methods of error corrected sequencing (ECS) that enable detection of very rare mutations well below the error rate of convention next generation sequencing (NGS). Further, the methods disclosed herein enable multiplex targeting of genomic DNA.

What is claimed is: 
     
         1 . A method of identifying a genetic mutation in a biological sample comprising nucleic acid obtained from a subject, the method comprising:
 a) amplifying one or more regions of interest from the biological sample comprising nucleic acid, wherein a plurality of amplicons for each region of interest are generated;   b) attaching an adapter and a random component to each amplicon generated in (a) and amplifying;   c) sequencing the amplicons comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified; and   d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation.   
     
     
         2 . The method of  claim 1 , wherein the biological sample comprises about 400 to about 800 ng nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein one region of interest is amplified in step (a). 
     
     
         4 . The method of  claim 1 , wherein more than one region of interest is amplified in step (a). 
     
     
         5 . The method of  claim 4 , wherein the regions of interest are amplified by PCR for about 15 to about 20 cycles generating a plurality of amplicons, the amplicons are separated into more than one reaction vial, one primer pair is added to each reaction vial, and the region of interest in each reaction vial and amplified for about 15 to about 20 cycles. 
     
     
         6 . The method of  claim 1 , wherein step (b) further comprises attaching an index sequence. 
     
     
         7 . The method of  claim 6 , wherein the adapter, the random component and the index sequence are attached via ligation. 
     
     
         8 . The method of  claim 1 , wherein the adapter is a Y-shaped adapter. 
     
     
         9 . The method of  claim 1 , wherein the adapter is an IIlumina adapter. 
     
     
         10 . The method of  claim 1 , wherein the adaptor and the random component are attached via ligation. 
     
     
         11 . The method of  claim 1 , wherein the method identifies clinical silent single-nucleotide variations (SNVs). 
     
     
         12 . The method of  claim 1 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 1,000 in the sample. 
     
     
         13 . The method of  claim 1 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 5,000 in the sample. 
     
     
         14 . The method of  claim 1 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 10,000 in the sample. 
     
     
         15 . A method of identifying a genetic mutation in a biological sample comprising nucleic acid obtained from a subject, the method comprising:
 a) hybridizing a primer pool comprising one or more primer pairs specific to one or more regions of interest from the biological sample comprising nucleic acid, extending from an upstream primer of the primer pair to a downstream primer of the primer pair, and ligating the extension product to the downstream primer of the primer pair, wherein products comprising the regions of interest flanked by sequences required for amplification are generated;   b) attaching an adapter comprising a random component and attaching an adapter comprising an index sequence to the products from (a) and amplifying;   c) sequencing the products comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified; and   d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation.   
     
     
         16 . The method of  claim 15 , wherein the biological sample comprises about 400 to about 800 ng nucleic acid. 
     
     
         17 . The method of  claim 15 , wherein more than 500 regions of interest are sequenced. 
     
     
         18 . The method of  claim 15 , wherein unbound primers are washed away prior to proceeding to (b). 
     
     
         19 . The method of  claim 15 , wherein the adapter is a Y-shaped adapter. 
     
     
         20 . The method of  claim 15 , wherein the adapter is an Illumina adapter. 
     
     
         21 . The method of  claim 15 , wherein the adapter comprising a random component and the adaptor comprising an index sequence are attached via PCR. 
     
     
         22 . The method of  claim 15 , wherein the method identifies clinical silent single-nucleotide variations (SNVs). 
     
     
         23 . The method of  claim 15 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 1,000 in the sample. 
     
     
         24 . The method of  claim 15 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 5,000 in the sample. 
     
     
         25 . The method of  claim 15 , wherein the method identifies a genetic mutation that is at a frequency of less than 1 in 10,000 in the sample. 
     
     
         26 . A method of detecting minimal residual disease (MRD) in a subject, the method comprising:
 a) hybridizing a primer pool comprising one or more primer pairs specific to one or more regions of interest from a biological sample comprising nucleic acid obtained from the subject, extending from an upstream primer of the primer pair to a downstream primer of the primer pair, and ligating the extension product to the downstream primer of the primer pair, wherein products comprising the regions of interest flanked by sequences required for amplification are generated;   b) attaching an adapter comprising a random component and attaching an adapter comprising an index sequence to the products from (a) and amplifying;   c) sequencing the products comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified; and   d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation and is indicative of MRD.   
     
     
         27 . The method of  claim 26 , wherein the subject is treated if a genetic mutation is detected.