Length and GC-Biases During Sequencing Library Amplification: A Comparison of Various Polymerase-Buffer Systems with Ancient and Modern DNA Sequencing Libraries
Open accessAbstract
High-throughput sequencing technologies frequently necessitate the use of PCR for sequencing library amplification. PCR is a sometimes enigmatic process and is known to introduce biases. Here we perform a simple amplification-sequencing assay using 10 commercially available polymerase-buffer systems to amplify libraries prepared from both modern and ancient DNA. We compare the performance of the polymerases with respect to a previously uncharacterized template length bias, as well as GC-content bias, and find that simply avoiding certain polymerase can dramatically decrease the occurrence of both. For amplification of ancient DNA, we found that some commonly used polymerases strongly bias against amplification of endogenous DNA in favor of GC-rich microbial contamination, in our case reducing the fraction of endogenous sequences to almost half.
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GC content of individual sequences affects PCR amplification efficiency in biological DNA.
“GC content of individual sequences were previously found to affect PCR amplification efficiency in biological DNA15–17.”
From Quantifying Molecular Bias in DNA Data Storage (2019) · cites this paper as [16]
Previous studies observed PCR bias in genomic biological sample amplification.
“Although previous studies observed PCR bias in genomic biological sample amplification15–17, it remained unclear whether such bias is significant in DNA data storage.”
From Quantifying Molecular Bias in DNA Data Storage (2019) · cites this paper as [16]
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- Authors
- Dabney, J., & Meyer, M.
- Affiliations
- Max Planck Society (DE)
- Venue
- BioTechniques
- Publisher
- Informa UK Limited
- Published
- 2012-02
- Volume
- 52
- Issue
- 2
- Pages
- 87-94
- ISSN
- 0736-6205 (ISSN-L)
- 1940-9818
- OpenAlex
- W1999518415
- Cited by
- 316
- References
- 27
- Citations by year
- 2026: 12 · 2025: 22 · 2024: 12 · 2023: 14 · 2022: 20 · 2021: 30 · 2020: 27 · 2019: 22 · 2018: 28 · 2017: 32 · 2016: 27 · 2015: 23 · 2014: 26 · 2013: 17 · 2012: 3
- FWCI
- 8.86
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- BioTechniques (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- Max Planck Society
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Keywords
- PCR bias, GC content bias, ancient DNA, microbial contamination, endogenous DNA, PCR amplification
- MeSH terms
- Base Composition, Buffers, DNA-Directed DNA Polymerase, Humans, Gene Library, Sequence Analysis, DNA, Nucleic Acid Amplification Techniques
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