Safe Sequencing System

Abstract

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

We claim: 
     
         1 . A method to analyze DNA using endogenous unique identifier sequences (UIDs), comprising:
 attaching adapter oligonucleotides to ends of fragments of analyte DNA of between 30 to 2000 bases, inclusive, to form adapted fragments, wherein each end of a fragment before said attaching is an endogenous UID for the fragment;   amplifying the adapted fragments using primers complementary to the adapter oligonucleotides to form families of adapted fragments;   determining nucleotide sequence of a plurality of members of a family; comparing nucleotide sequences of the plurality of members of the family; and   identifying a nucleotide sequence as accurately representing an analyte DNA fragment when at least 1% of members of the family contain the sequence.   
     
     
         2 . The method of  claim 1  further comprising:
 enriching for fragments representing one or more selected genes by means of capturing a subset of the fragments using capture oligonucleotides complementary to selected genes in the analyte DNA. 
 
     
     
         3 . The method of  claim 1  further comprising:
 enriching for fragments representing one or more selected genes by means of amplifying fragments complementary to selected genes. 
 
     
     
         4 . The method of  claim 2  wherein the step of attaching is prior to the step of enriching. 
     
     
         5 . The method of  claim 3  wherein the step of attaching is prior to the step of enriching. 
     
     
         6 . The method of  claim 1  wherein the fragments are formed by shearing. 
     
     
         7 . The method of  claim 1  wherein a nucleotide sequence is identified as accurately representing an analyte DNA fragment when at least 5% of members of the family contain the sequence. 
     
     
         8 . The method of  claim 1  wherein prior to the amplification, the analyte DNA is treated with bisulfite to convert unmethylated cytosine bases to uracil. 
     
     
         9 . The method of  claim 1  further comprising the step of comparing number of families representing a first analyte DNA fragment to number of families representing a second analyte DNA fragment to determine a relative concentration of a first analyte DNA fragment to a second analyte DNA fragment in the plurality of analyte DNA fragments.