The DOI matches its Crossref record; 34 of the 34 references it cites were found in a registry or an index.
Sources of PCR-induced distortions in high-throughput sequencing data sets
Open accessPublished version of a preprint
Abstract
PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.
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Cites
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Removing Noise From Pyrosequenced Amplicons
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Self-citationOverview of methodologies for T-cell receptor repertoire analysis
Rosati, E., Dowds, C. M., Liaskou, E., Henriksen, E. K. K., Karlsen, T. H., & Franke, A. (2017) · BMC Biotechnology · doi:10.1186/s12896-017-0379-9 · Cited by 322
Singh, M., Al-Eryani, G., Carswell, S., Ferguson, J. M., Blackburn, J., Barton, K., Roden, D., Luciani, F., Giang Phan, T., Junankar, S., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11049-4 · Cited by 308
Quantifying Molecular Bias in DNA Data Storage
Chen, Y.-J., Takahashi, C. N., Organick, L., Stewart, K., Ang, S. D., Weiss, P., Peck, B., Seelig, G., Ceze, L., & Strauss, K. (2019) · doi:10.1101/566554 · Cited by 116
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Metadata
- Authors
- Kebschull, J. M., & Zador, A. M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2015-07-17
- Volume
- 43
- Issue
- 21
- Pages
- gkv717
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2131176533
- Cited by
- 299
- References
- 34
- FWCI
- 6.82
- Type
- journal article
- Language
- en
- Open access
- gold
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