Sources of PCR-induced distortions in high-throughput sequencing data sets

Kebschull, Justus M.; Zador, Anthony M.

doi:10.1093/nar/gkv717

Cited by 218 publications

(239 citation statements)

References 32 publications

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“…Prior to sequencing, many sample preparation workflows employ DNA amplification, particularly the Polymerase Chain Reaction (PCR). Consequently, errors that arise during PCR lead to false positive mutations and can obscure sequencing results, making it especially challenging to identify rare genetic variation [2]. In addition, the inherently high error rate of next-generation sequencing technologies requires additional steps to distinguish true mutation events from sequencing errors and is an active area of research [3–6].…”

Section: Introductionmentioning

confidence: 99%

“…Studies have also shown that recombinants can also arise between complementary strands, or even during a single round of polymerase extension [22]. The rate of PCR-mediated recombination by polymerases has been studied by a phenotypic assay and next-generation sequencing, but has yet to be directly assayed by single-molecule sequencing methods [2, 23, 24]. …”

Section: Introductionmentioning

confidence: 99%

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Examining Sources of Error in PCR by Single-Molecule Sequencing

2017

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Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Examining Sources of Error in PCR by Single-Molecule Sequencing

2017

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“…For instance, template concentration, bias against high GC templates, template switching, and polymerase errors may contribute to errors in downstream steps [27–29]. These previous studies indicated that these bias and errors can be minimized by using the minimum number of amplification cycles required to form products and defining the optimal template concentration in the PCR reaction.…”

Section: Resultsmentioning

confidence: 99%

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Niland

Jankowsky

Harris

2016

Analytical Biochemistry

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Quantification of the specificity of RNA binding proteins and RNA processing enzymes is essential to understanding their fundamental roles in biological processes. High Throughput Sequencing Kinetics (HTS-Kin) uses high throughput sequencing and internal competition kinetics to simultaneously monitor the processing rate constants of thousands of substrates by RNA processing enzymes. This technique has provided unprecedented insight into the substrate specificity of the tRNA processing endonuclease ribonuclease P. Here, we investigate the accuracy and robustness of measurements associated with each step of the HTS-Kin procedure. We examine the effect of substrate concentration on the observed rate constant, determine the optimal kinetic parameters, and provide guidelines for reducing error in amplification of the substrate population. Importantly, we find that high-throughput sequencing, and experimental reproducibility contribute their own sources of error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed.

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“…Differences in hybridization efficiency during capture, due to GC content variation in the genome, account for the poor coverage of some target exon regions. DNA regions with nucleotide repeats may also result in coverage problems (19, 20). Pseudogenes, as for IKBKG and NCF1 , also hamper correct data generation.…”

Section: Generating Next Generation Sequencing Datamentioning

confidence: 99%

Exome and genome sequencing for inborn errors of immunity

Meyts

Bosch

Bolze

et al. 2016

Journal of Allergy and Clinical Immunology

170

148

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The advent of next generation sequencing (NGS) in 2010 has transformed medicine and particularly the growing field of monogenic inborn errors of immunity, including primary immunodeficiencies (PID). NGS has facilitated the discovery of novel disease-causing genes and the genetic diagnosis of patients with PID. Whole-exome sequencing (WES) is presently the most cost-effective approach for PID research and diagnostics, though whole genome sequencing (WGS) offers several advantages. The scientific or diagnostic challenge consists in selecting one or two candidate variants among thousands of NGS calls. Variant- and gene-level computational methods as well as immunological hypotheses can help to narrow down this genome-wide search. The key to success is a well-informed genetic hypothesis on three key aspects: mode of inheritance, clinical penetrance, and genetic heterogeneity of the condition. This determines the search strategy and the frequency cut-offs for candidate alleles. Subsequent functional validation of the disease-causing effect of the candidate variant is critical. Even the most up-to-date dry lab cannot clinch this validation without a seasoned wet lab. The multifariousness of variations entails an experimental rigor even greater than traditional Sanger sequencing-based approaches, in order not to assign PID to false positives. Finding the needle in the haystack takes patience, prudence, and discernment.

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Sources of PCR-induced distortions in high-throughput sequencing data sets

Cited by 218 publications

References 32 publications

Examining Sources of Error in PCR by Single-Molecule Sequencing

Examining Sources of Error in PCR by Single-Molecule Sequencing

Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates

Exome and genome sequencing for inborn errors of immunity

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