Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells
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- dc.contributor.author Vonesch, Sibylle C.
- dc.contributor.author Li, Shengdi
- dc.contributor.author Szu-Tu, Chelsea
- dc.contributor.author Henning, Bianca P.
- dc.contributor.author Dobrev, Nikolay
- dc.contributor.author Steinmetz, Lars M.
- dc.date.accessioned 2022-05-03T10:28:57Z
- dc.date.available 2022-05-03T10:28:57Z
- dc.date.issued 2021
- dc.description.abstract Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to other organisms.
- dc.description.sponsorship This work was supported by an Advanced Investigator grant from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (AdG-742804—SystGeneEdit to L.M.S.). S.C.V. was supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation (grant number P300PA_177909)
- dc.format.mimetype application/pdf
- dc.identifier.citation Vonesch SC, Li S, Szu Tu C, Henning BP, Dobrev N, Steinmetz LM. Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells. G3 (Bethesda). 2021 Jan 18;11(1):jkaa009. DOI:10.1093/g3journal/jkaa009
- dc.identifier.doi http://dx.doi.org/10.1093/g3journal/jkaa009
- dc.identifier.issn 2160-1836
- dc.identifier.uri http://hdl.handle.net/10230/52968
- dc.language.iso eng
- dc.publisher Oxford University Press
- dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/742804
- dc.rights © Sibylle C Vonesch et al. 2020. Published by Oxford University Press on behalf of Genetics Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
- dc.rights.accessRights info:eu-repo/semantics/openAccess
- dc.rights.uri http://creativecommons.org/licenses/by/4.0/
- dc.subject.other Genòmica
- dc.subject.other ADN
- dc.title Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells
- dc.type info:eu-repo/semantics/article
- dc.type.version info:eu-repo/semantics/publishedVersion