Somatic mutation landscapes at single-molecule resolution

Abascal, Federico; Harvey, Luke M. R.; Mitchell, Emily; Lawson, Andrew; Lensing, Stefanie; Ellis, Peter; Russell, Andrew J.; Alcantara, Raul E.; Baez‐Ortega, Adrian; Wang, Yichen; Kwa, Eugene Jing; Lee-Six, Henry; Cagan, Alex; Coorens, Tim; Chapman, Michael Spencer; Ólafsson, Sigurgeir; Leonard, Steven; Jones, David R.; Machado, Heather E.; Davies, Megan; Øbro, Nina Friesgaard; Mahubani, Krishnaa T.; Allinson, Kieren; Gerstung, Moritz; Saeb‐Parsy, Kourosh; Kent, David G.; Laurenti, Elisa; Stratton, Michael R.; Rahbari, Raheleh; Campbell, Peter J.; Osborne, Robert J.; Martincorena, Iñigo

doi:10.1038/s41586-021-03477-4

Cited by 320 publications

(413 citation statements)

References 58 publications

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“…While multiple S protein mutations, i.e. B.1.1.7 variant, have been associated with the severity of COVID-19 [ 17 , 18 ], this is not the case for our patients. Our samples did not consist of B.1.1.7 variant.…”

Section: Discussioncontrasting

confidence: 55%

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Molecular epidemiology of SARS-CoV-2 isolated from COVID-19 family clusters

et al. 2021

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Background Transmission within families and multiple spike protein mutations have been associated with the rapid transmission of SARS-CoV-2. We aimed to: (1) describe full genome characterization of SARS-CoV-2 and correlate the sequences with epidemiological data within family clusters, and (2) conduct phylogenetic analysis of all samples from Yogyakarta and Central Java, Indonesia and other countries. Methods The study involved 17 patients with COVID-19, including two family clusters. We determined the full-genome sequences of SARS-CoV-2 using the Illumina MiSeq next-generation sequencer. Phylogenetic analysis was performed using a dataset of 142 full-genomes of SARS-CoV-2 from different regions. Results Ninety-four SNPs were detected throughout the open reading frame (ORF) of SARS-CoV-2 samples with 58% (54/94) of the nucleic acid changes resulting in amino acid mutations. About 94% (16/17) of the virus samples showed D614G on spike protein and 56% of these (9/16) showed other various amino acid mutations on this protein, including L5F, V83L, V213A, W258R, Q677H, and N811I. The virus samples from family cluster-1 (n = 3) belong to the same clade GH, in which two were collected from deceased patients, and the other from the survived patient. All samples from this family cluster revealed a combination of spike protein mutations of D614G and V213A. Virus samples from family cluster-2 (n = 3) also belonged to the clade GH and showed other spike protein mutations of L5F alongside the D614G mutation. Conclusions Our study is the first comprehensive report associating the full-genome sequences of SARS-CoV-2 with the epidemiological data within family clusters. Phylogenetic analysis revealed that the three viruses from family cluster-1 formed a monophyletic group, whereas viruses from family cluster-2 formed a polyphyletic group indicating there is the possibility of different sources of infection. This study highlights how the same spike protein mutations among members of the same family might show different disease outcomes.

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Section: Discussioncontrasting

confidence: 55%

“…These variants might be associated with some potential advantages for these viruses. While the B.1.1.7 variant has been associated with COVID-19 clinical severity [ 17 , 18 ], the 501.V2 and P.1 variants have not [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of SARS-CoV-2 isolated from COVID-19 family clusters

et al. 2021

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“…Future strategies may involve digesting single-stranded DNA irrespective of whether it contains a recognizable lesion, or labelling resynthesized bases and excluding from analysis. Noteworthily, Abascal et al recently reported nanorate sequencing (NanoSeq) that suppresses strand resynthesis during ER/AT by using a restriction enzyme to digest intact DNA to produce blunted dsDNA fragments and then non-A dideoxynucleotides during dA-tailing to block templated extension 31 . As a result, NanoSeq can achieve a reported error rate of < 5e-9 when applied to gDNA extracted from sperm and cord blood samples.…”

Section: Discussionmentioning

confidence: 99%

Duplex-Repair enables highly accurate sequencing, despite DNA damage

Xiong

Shea

Rhoades

et al. 2021

Preprint

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Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via End Repair/dA-Tailing, may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior duplex base pairs from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles heel in sequencing and offers a solution to restore high accuracy.

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“…Unique molecular identifiers (UMIs) have been developed to suppress the errors to detect mutations below 0.1% VAF 19,20 . Recent advances in DuplexSeq 21 , NanoSeq 22 and SaferSeqS 23 has further reduced errors by grouping both strands of a DNA molecule together into a duplex family to distinguish DNA damage with real mutation achieving confident variant calling at 0.01% VAF or lower. However, since all template molecules, regardless of wild type or variant molecules, are sequenced redundantly in current UMI-based methods, they require sequencing to extremely high depths proportional to input molecule amount.…”

Section: Introductionmentioning

confidence: 99%

“…We introduced different UMI sequences to each strand of DNA molecule by PCR and thus duplex family information is lost during denaturation of UMI attachment PCR. We expect that error from DNA damage may be further suppressed in QBDA by using ligation-based UMI attachment, so that in downstream bioinformatics analysis both strands of a DNA molecule can be grouped into a duplex family similar to DuplexSeq 21 , NanoSeq22 and SaferSeqS23 while still reducing sequencing depth by BDA variant enrichment. The gene ploidy impacts VAF in QBDA, but QBDA is able to accurately quantitate VAF in case CNV and mutation are simultaneously present in the gene of interest as long as copy number for the gene is normalized.…”

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confidence: 99%

Calibration-free NGS Quantitation of Mutations below 0.01% VAF

Dai

Chen

et al. 2021

Preprint

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The quantitation of rare somatic mutations is essential for basic research and translational clinical applications including minimal residual disease (MRD) detection. Though unique molecular identifier (UMI) has suppressed sequencing error and allowed detection rare mutation, the sequencing depth requirement is high. The blocker displacement amplification (BDA) allele enrichment method allows detection of rare mutations using low sequencing depth, but requires calibration to accurately quantitate the VAF of novel mutations. Here, we present Quantitative Blocker Displacement Amplification (QBDA), a method that allows accurate detection and quantitation of mutations below 0.01% VAF at only 23,000X depth. QBDA integrates sequence-selective variant enrichment into UMI quantitation allowing confident detection of rare mutations and reduced sequencing depth. Using a panel of 20 genes recurrently altered in acute myeloid leukemia, we demonstrate quantitation of various mutations including single base substitutions and indels down to a VAF of 0.001% at a single locus with less than 4 million sequencing reads, allowing a sensitive minimal residual disease (MRD) detection in patients during complete remission. In a comprehensive pan-cancer panel covering 61 genes and a melanoma hotspot panel covering 8 genes, we detect mutations down to 0.1% VAF using only 1 million reads in a broad range of clinical samples including cell-free DNA and FFPE DNA, enabling tissue or liquid biopsy genetic tests with de-centralized sequencing instruments. QBDA thus provides a convenient and versatile method for sensitive mutation quantitation using low-depth sequencing.

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Somatic mutation landscapes at single-molecule resolution

Cited by 320 publications

References 58 publications

Molecular epidemiology of SARS-CoV-2 isolated from COVID-19 family clusters

Molecular epidemiology of SARS-CoV-2 isolated from COVID-19 family clusters

Duplex-Repair enables highly accurate sequencing, despite DNA damage

Calibration-free NGS Quantitation of Mutations below 0.01% VAF

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