Accuracy of Next Generation Sequencing Platforms

Abstract

Next-generation DNA sequencing has revolutionized genomic studies and is driving the implementation of precision diagnostics. The ability of these technologies to disentangle sequence heterogeneity, however, is limited by their relatively high error rates. A Several single molecule barcoding strategies have been propose to reduce the overall error frequency. A Duplex Sequencing additionally exploits the fact that DNA is double-strand, with one strand reciprocally encoding the sequence information of its complement, and can eliminate nearly all sequencing errors by comparing the sequence of individually tagged amplicons derived from one strand of DNA with that of its complementary strand. This method reduces errors to fewer than one per ten million nucleotides sequenced.

Keywords: Accuracy; Duplex sequencing; Next-generation DNA sequencing; Precision medicine.

Figure 1

Comparison of the probability that an observed variant is real [54] for subclonal variants using Q30-filtered reads of an Illumina HiSeq2500 (NGS) versus Duplex Sequencing. Error Frequencies of each approach is given in parenthesis. PPV (Positive Predictive Value)=(Expected Number of true positives)/(Expected Total Number of Positive Calls). Note that the PPV is 0.50 for NGS when the variant frequency at a single position is ~1/100, i.e., any variant call has a 50/50 chance of being real hen the frequency of real variants equals the frequency of mistakes invalidity [62].