Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

Miner, Brooks E.; Stöger, Reinhard; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

doi:10.1093/nar/gnh132

Cited by 67 publications

(77 citation statements)

References 12 publications

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“…UIDs, sometimes called barcodes or indexes, can be assigned to nucleic acid fragments using a variety of methods. These methods include the introduction of exogenous sequences through PCR (40,41) or ligation (42,43). Even more simply, randomly sheared genomic DNA inherently contains UIDs consisting of the sequences of the two ends of each sheared fragment ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Detection and quantification of rare mutations with massively parallel sequencing

Kinde

Papadopoulos

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

1,045

973

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The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.diagnostics | early diagnosis | biomarkers | genetics | cancer

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Section: Resultsmentioning

confidence: 99%

Detection and quantification of rare mutations with massively parallel sequencing

Kinde

Papadopoulos

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

1,045

973

View full text Add to dashboard Cite

show abstract

“…The 440 bp band obtained upon electrophoresis in a 1.5% agarose gel was purified (QIAquick gel extraction kit, Qiagen), cloned (Invitrogen, TA Cloning Kit; E. coli, DH5α), and sequenced (Translational Genomic Research Institute, Phoenix, AZ). The sequencing revealed only four pairs of DNA clones with identical sequences, including in the degenerate linker region 65 out of 395 sequenced clones. These were eliminated from consideration.…”

Section: Methodsmentioning

confidence: 99%

Hemimethylation footprints of DNA demethylation in cancer

Shao¹,

Lacey²,

Dubeau³

et al. 2009

Epigenetics

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Hypomethylation of DNA repeats, including satellite 2 DNA (Sat2), is one of the most frequent epigenetic changes in cancer. We examined ovarian epithelial tumors and diverse control tissues for methylation on only one strand (hemimethylation), both strands (symmetrical methylation), or neither strand at Sat2 CpG dyads using hairpin genomic sequencing. Analysis of the resulting cloned DNA molecules indicated that although carcinomas displayed much symmetrical hypomethylation of CpG dyads, there was cancer-linked hypermethylation at one of the thirteen dyads in the examined 0.2 kb Sat2 region. Hemimethylated sites were seen in both carcinomas and controls but, importantly, in carcinoma DNA molecules, they were significantly more likely to occur in clusters displaying the same orientation (the same strand methylated). Our data suggest that hemimethylated CpG dyads are intermediates in active demethylation during carcinogenesis and not just due to a failure of maintenance methylation during replicative DNA synthesis. Constitutive heterochromatin may be especially suitable for providing a snapshot of demethylation intermediates because hemimethylation might be more long-lived in heterochromatin due to its highly condensed state.

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“…Single molecule tagging (SMT) is a method that has been developed to identify PCR duplicates using a DNA sequence tag [20][21][22] and has recently been incorporated into RNA sequencing [23]. Random oligomers are incorporated into a template prior to PCR amplification, and duplicates are tracked by the SMT oligomer.…”

Section: Introductionmentioning

confidence: 99%

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

et al. 2014

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Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

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Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

Cited by 67 publications

References 12 publications

Detection and quantification of rare mutations with massively parallel sequencing

Detection and quantification of rare mutations with massively parallel sequencing

Hemimethylation footprints of DNA demethylation in cancer

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

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