Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

Joseph B. Hiatt, Colin C. Pritchard, Stephen J. Salipante, Brian O'Roak, Jay Shendure

Research output: Contribution to journalArticle

168 Citations (Scopus)

Abstract

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10-6 in cell lines and 2.6 × 10-5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p. V600E (melanoma, 0.2% alternate allele frequency), KRAS p. G12V (lung, 0.6%), JAK2 p. V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p. Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.

Original languageEnglish (US)
Pages (from-to)843-854
Number of pages12
JournalGenome Research
Volume23
Issue number5
DOIs
StatePublished - May 2013
Externally publishedYes

Fingerprint

Molecular Probes
Cell Line
Mutation
Melanoma
Colon
High-Throughput Nucleotide Sequencing
Lung
Deamination
Genetic Heterogeneity
Neoplasm Genes
Cytosine
Medical Genetics
Guanine
Mutation Rate
Point Mutation
Gene Frequency
DNA Damage
Neoplasms
Costs and Cost Analysis
DNA

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. / Hiatt, Joseph B.; Pritchard, Colin C.; Salipante, Stephen J.; O'Roak, Brian; Shendure, Jay.

In: Genome Research, Vol. 23, No. 5, 05.2013, p. 843-854.

Research output: Contribution to journalArticle

Hiatt, Joseph B. ; Pritchard, Colin C. ; Salipante, Stephen J. ; O'Roak, Brian ; Shendure, Jay. / Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. In: Genome Research. 2013 ; Vol. 23, No. 5. pp. 843-854.
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