Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes
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Abstract
Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.
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Variant sequences were verified by next-generation sequencing and manual inspection.
“To verify the sequences of variants, next-generation sequencing libraries were prepared according to a low-volume Nextera sequencing protocol 43 and sequenced on the Illumina Miseq platform (single-end mode with 50 bp R1).”
From Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2 (2022) · cites this paper as [43]
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- Authors
- Baym, M., Kryazhimskiy, S., Lieberman, T. D., Chung, H., Desai, M. M., & Kishony, R.
- Affiliations
- Harvard University (US)
- Corresponding author
- Baym, Michael
- Kryazhimskiy, Sergey
- Lieberman, Tami D.
- Chung, Hattie
- Desai, Michael M.
- Kishony, Roy
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-05-22
- Volume
- 10
- Issue
- 5
- Pages
- e0128036
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4441430
- OpenAlex
- W2136678168
- Cited by
- 685
- References
- 18
- Citations by year
- 2026: 35 · 2025: 54 · 2024: 72 · 2023: 64 · 2022: 92 · 2021: 105 · 2020: 89 · 2019: 63 · 2018: 47 · 2017: 34 · 2016: 22 · 2015: 7 · 2014: 1
- FWCI
- 16.06
- Type
- journal article
- Language
- en
- Update notices
- correction (2015-06-18)
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Science Foundation (R01-GM104239, 0903013, 1313638, GM104239)
- Alfred P. Sloan Foundation (GM104239, PHY 1313638)
- James S. McDonnell Foundation (PHY 1313638, GM104239)
- National Institutes of Health (PHY 1313638, 281891)
- National Institute of General Medical Sciences (R01GM081617, R01GM104239)
- Division of Mathematical Sciences
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genetics, Bioinformatics, and Biomedical Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- small genomes, sample pooling, experimental evolution, genetic screen, environmental samples, artificial chromosomes, RNA sequencing
- MeSH terms
- Gene Library, Sequence Analysis, DNA, Genomics, High-Throughput Nucleotide Sequencing
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