Tag Archives: Duplex Sequencing

A Method to Increase Accuracy in Next Generation Sequencing

B) Duplex Sequencing workflow. Sheared, T-tailed double-stranded DNA is ligated to A-tailed adapters. Because every adapter contains<br />a Duplex Tag on each end, every DNA fragment becomes labeled with two distinct tag sequences (arbitrarily designated α and β in the single fragment shown).<br />PCR amplificationwith primers containing Illumina flow-cell–compatible tails is carried out to generate families of PCR duplicates. Two types of PCR products are<br />produced from each DNA fragment. Those derived from one strand will have the α tag sequence adjacent to flow cell sequence 1 and the β tag sequence<br />adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally.

Next-generation sequencing allows detection of minor variants in a heterogeneous sample. However, errors in PCR and sequencing pose limits on its sensitivity.
A group at University of Washington developed a method, called Duplex Sequencing, to dramatically improve accuracy by sequencing both strands of each DNA duplex. Mutations that are detected in the consensus sequence of one strand but not the other are discounted as technical errors.

The authors adopted the method to Illumina sequencing. It involves the use of modified adaptors that have a tag with random sequence attached. After ligation of these modified adaptors, each duplex DNA fragment is flanked by two different tags and subjected to paired-end sequencing. Sequences of the same duplex from the complementary strands can therefore be uniquely identified by having the same tags on either ends. Comparing sequences of the two strands allows identification of true mutations. The authors estimated that Duplex sequencing has a theoretical background error rate of less than one per 109 nucleotides sequenced.
Full text article can be accessed here: http://www.pnas.org/content/early/2012/07/31/1208715109.full.pdf