Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair-derived artifacts as residual errors.
Arch Toxicol
; 94(10): 3475-3485, 2020 10.
Article
em En
| MEDLINE
| ID: mdl-32737516
ABSTRACT
To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strand-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 7 bp of the library fragments in the 5'-NpCpA-3' and 5'-NpCpT-3' trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 7 bp, PECC-Seq could reduce the sequencing error frequency to mid-10-7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10-8. In mutagen-treated (6 µg/mL methyl methanesulfonate or 12 µg/mL N-nitroso-N-ethylurea) TK6, increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights into further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.
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Base de dados:
MEDLINE
Assunto principal:
Análise Mutacional de DNA
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Sequenciamento de Nucleotídeos em Larga Escala
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Taxa de Mutação
Tipo de estudo:
Diagnostic_studies
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Guideline
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Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article