Detection and quantification of rare mutations with massively parallel sequencing

Kinde, Isaac; Wu, Jian; Papadopoulos, Nick; Kinzler, Kenneth W.; Vogelstein, Bert

doi:10.1073/pnas.1105422108

Cited by 1,065 publications

(1,012 citation statements)

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“…Its reliability results from an overdispersed statistical model of nucleotide counts and from integrating the signals from both DNA strands. The current limit of detection is around 1/10,000 alleles, but it may be further improved by increased coverage and higher sequencing fidelity with improved biochemistry or barcoded reads 21 .…”

Section: Discussionmentioning

confidence: 99%

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

et al. 2012

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According to the clonal evolution model, tumour growth is driven by competing subclones in somatically evolving cancer cell populations, which gives rise to genetically heterogeneous tumours. Here we present a comparative targeted deep-sequencing approach combined with a customised statistical algorithm, called deepsnV, for detecting and quantifying subclonal single-nucleotide variants in mixed populations. We show in a rigorous experimental assessment that our approach is capable of detecting variants with frequencies as low as 1/10,000 alleles. In selected genomic loci of the TP53 and VHL genes isolated from matched tumour and normal samples of four renal cell carcinoma patients, we detect 24 variants at allele frequencies ranging from 0.0002 to 0.34. moreover, we demonstrate how the allele frequencies of known single-nucleotide polymorphisms can be exploited to detect loss of heterozygosity. our findings demonstrate that genomic diversity is common in renal cell carcinomas and provide quantitative evidence for the clonal evolution model.

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Section: Discussionmentioning

confidence: 99%

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

et al. 2012

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“…Furthermore, advances in whole genome amplification and nextgeneration sequencing from single-cells have enabled detection of rare molecular events in clinical specimens and are also promising methods for biomarker assays. [23][24][25] Unlike conventional MethyLight PCR, the droplets in MethyLight ddPCR are cycled to endpoint and each droplet is read as positive or negative. 15 Thus, amplification efficiency is less of a concern in MethyLight ddPCR compared to conventional MethyLight PCR.…”

Section: Discussionmentioning

confidence: 99%

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Carter

Makar

et al. 2015

Epigenetics

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“…Illumina sequencing runs consistently display a base-pair error rate of 0.05 to 1%[115]. The di-base encoding used by the SOLiD machine is able to lower this error rate to about 0.075% (SOLiD application note).…”

Section: Mps Applications In Mutation Analysismentioning

confidence: 99%

“…This can only be done with errors that exhibit some consistent patterns related to the library preparation and sequencing steps[116] and not feasible for errors that appear at random. Interestingly, a recent approach to the identification of rare variants utilized the PCR amplification step in the (Illumina) library preparation to identify families of templates carrying the same mutation, which then could be inferred to have been present in the original template molecule as a real mutation[115]…”

Section: Mps Applications In Mutation Analysismentioning

confidence: 99%

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5,000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.

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Detection and quantification of rare mutations with massively parallel sequencing

Cited by 1,065 publications

References 50 publications

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

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