Sequencing methods and kits

Abstract

A method for detecting rare genomic variants in a population of cells is disclosed. The method can detect de novo mutations in bacteria and analyze the impact of various physiological conditions on mutation rate, even though such effects would be too subtle to detect using other methods. The method can be used for detection of low-frequency subpopulations in the microbiome or in cancer.

1 - 7 . (canceled) 
     
     
         8 . A method for sequencing mutations in the genome of an organism, comprising the steps of:
 (a) cleaving a genomic DNA that contains one or more regions of interest (ROI), wherein the genomic DNA is cleaved at opposing locations relative to the one or more ROI;   (b) ligating forked adapters to opposite 3′ ends of the genomic DNA, wherein the forked adapters comprise a barcode DNA sequence;   (c) forming a linear amplification mixture comprising the genomic DNA;   (d) annealing adapter barcode primers to the 3′ end of the forked adapters within the linear amplification mixture, and performing one cycle of linear amplification with at least one of the adapter barcoded primers to yield a single round linear amplification product;   (e) performing N cycles of the linear amplification with forward adapter amplifier primers; and   (f) performing exponential PCR with the forward adapter amplifier primers, the adapter-reverse-primers and reverse adapter amplifier primers to produce a polymerase chain reaction (“PCR”) products suitable for DNA sequencing.   
     
     
         9 . The method of  claim 8 , further comprising the step of aligning the sequences obtained in step (f) to quantify mutations in the ROI. 
     
     
         10 . The method of  claim 8 , wherein the genomic DNA of the organism is cleaved by digestion with a restriction enzyme. 
     
     
         11 . The method of  claim 8 , further comprising the step of removing unused barcode primers from the single round linear amplification product. 
     
     
         12 . The method of  claim 8 , wherein exponential PCR is performed by adding an adapter reverse primer and a reverse adapter primer to the linear amplification product. 
     
     
         13 . The method of  claim 8 , wherein the adapter barcoded primer comprises an adapter region, a barcode region and an annealing region. 
     
     
         14 . The method of  claim 8 , wherein the forward adapter amplifier primer shares a region of homology with an adapter region of the adapter barcoded primer. 
     
     
         15 . A kit for performing a method for sequencing DNA in the genome of an organism, the method comprising:
 (a) digesting genomic DNA of the organism with an enzyme that cleaves at the 3′ end of a region of interest (ROI) to produce digested genomic DNA;   (b) forming a mixture comprising the digested genomic DNA and an adapter barcoded primer, wherein the adapter barcoded primer comprises, from 5′ to 3′, an adapter region, a barcode region and an annealing region that anneals at the 3′ end of the ROI and performing a single round of extension using the mixture to produce a single round extension product containing a strand comprising, from 5′ to 3′, the ROI, a first sequence that is complementary to the barcode region and is attached to the 3′ end of the ROI, and a second sequence complementary to the adapter region;   (c) performing N cycles of linear amplification using the strand obtained in (b) as a template and a forward adapter amplifier primer to produce a linear amplification product;   (d) performing exponential polymerase chain reaction (PCR) with the linear amplification product to produce an amplified product using at least two primers; and   (e) sequencing the amplified product,   wherein the kit comprises adapter barcode primers, forward adapter amplifier primers, adapter-reverse-primers and reverse adapter amplifier primers for a region of interest in the genome of an organism.   
     
     
         16 . A kit for performing the method of  claim 8 , comprising forked adapters, adapter barcode primers, forward adapter amplifier primers, adapter-reverse-primers and reverse adapter amplifier primers for a region of interest in the genome of an organism. 
     
     
         17 . The kit of  claim 16 , wherein the forward adapter amplifier primer shares a region of homology with the adapter region of the adapter barcoded primer. 
     
     
         18 . A kit for performing the method of  claim 8 , comprising forked adapters, adapter barcode primers, forward adapter amplifier primers, adapter-reverse-primers and reverse adapter amplifier primers for a region of interest in the genome of an organism. 
     
     
         19 . The kit of  claim 18 , wherein the adapter barcoded primer comprises an adapter region, a barcode region and an annealing region. 
     
     
         20 . The kit of  claim 19 , wherein the forward adapter amplifier primer shares a region of homology with the adapter region of the adapter barcoded primer.