Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates.
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| - Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates. (en)
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| - Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates. This method uses degenerate molecular tags in addition to sequencing adapters to recognize reads originating from each strand of DNA. The generated sequencing reads then will be analyzed using two methods: single-strand consensus sequences (SSCS) and duplex consensus sequences (DCS) assembly. Duplex sequencing theoretically can detect mutations with frequencies as low as 5 x 10ā8 --that is more than 10,000 times higher in accuracy compared to the conventional next-generation sequencing methods. The estimated error rate of standard next-generation sequencing platforms is 10ā2 to 10ā3 per base call. With this error rate, billions of base calls that are produced by NGS will result in millions of errors. The errors are introduced during sample preparation and sequencing such as polymerase chain reaction, sequencing, and image analysis errors. While the NGS platforms' error rate is acceptable in some applications such as detection of clonal variants, it is a major limitation for applications that require higher accuracy for detection of low-frequency variants such as detection of intra-organismal mosaicism, subclonal variants in genetically heterogeneous cancers, or circulating tumor DNA. Several library preparation strategies have been developed that increase accuracy of NGS platforms such as molecular barcoding and circular consensus sequencing method. Like NGS platforms, the data generated by these methods originates from a single strand of DNA, and therefore the errors that are introduced during PCR amplification, tissue processing, DNA extraction, hybridization capture (where used) or DNA sequencing itself can still be distinguished as a true variant. The duplex sequencing method addresses this problem by taking advantage of the complementary nature of two strands of DNA and confirming only variants that are present in both strands of DNA. Because the probability of two complementary errors arising at the same location in both strands is exceedingly low, duplex sequencing increases the accuracy of sequencing significantly. (en)
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