Single duplex DNA sequencing with CODEC detects mutations with high sensitivity

Abstract

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules (‘single duplexes’) to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10−8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Error-corrected deep targeted sequencing of circulating cell-free DNA from colorectal cancer patients for sensitive detection of circulating tumor DNA

“…Similar to other studies, UMIseq enabled ctDNA detection down to a cAF below 10 -4 with the lowest observed cAF in a ctDNA positive sample being 0.004% and a theoretical LOD below 0.001% in 10% of the estimated LODs at 20,000x sequencing depth. Similar to findings in other studies 13,21,22 , the error rate of UMIseq was highly dependent on nucleotide substitution and trinucleotide context. Particularly, C>T substitutions in the context of N(C>T)G were error-prone, while T>G and C>G substitutions, regardless of trinucleotide context, had a low error rate.…”

“…Similar to findings in other studies 13,21,22 , the error rate of UMIseq was highly dependent on nucleotide substitution and trinucleotide context. Particularly, C>T substitutions in the context of N(C>T)G were error-prone, while T>G and C>G substitutions, regardless of trinucleotide context, had a low error rate.…”

“…This has multiple weaknesses e.g., that plasma volumes, sample processing, and cohort compositions are different. The analytical sensitivities observed in plasma samples are, a bit surprisingly, quite similar for targeted and WGS approaches -often the lowest detected AFs are in the range 10 -4 to 10 -5 , indicating that in practice both targeted sequencing and WGS strategies have their own limitations 11,12,14,20,21 .…”

“…, that plasma volumes, sample processing, and cohort compositions are different. The analytical sensitivities observed in plasma samples are, a bit surprisingly, quite similar for targeted and WGS approaches - often the lowest detected AFs are in the range 10 -4 to 10 -5 , indicating that in practice both targeted sequencing and WGS strategies have their own limitations 11,12,14,20,21 . Presumably, targeted strategies that employ comprehensive error correction through UMIs or Duplex sequencing are limited by the amount of signal captured for ctDNA detection, while WGS approaches are limited by their error rate despite comprehensive error modeling and correction.…”

Multi-gene measurable residual disease assessed by digital polymerase chain reaction has clinical and biological utility in acute myeloid leukemia patients receiving venetoclax/azacitidine

“…This could have impacted our results and led to misclassification of patients as MRD-negative who actually had residual disease. An ultra-sensitive NGS platform such as those recently described 6,8,41,42 is another alternative for molecular MRD monitoring, although currently these platforms are more labor-intensive and more costly than ddPCR. Consortium efforts to standardize molecular MRD for future studies are underway and will be essential to the establishment of this modality for clinical use.…”