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.
Highlights
Whole-genome sequencing is a powerful tool in genomics research by providing an unbiased and comprehensive view of the genetic alterations present in a cell
Direct preparation of sequencing libraries from yeast cultures without DNA isolation enables massively scaled genome sequencing experiments that have so far been hampered by the time and effort spent on genomic DNA isolation
Initial comparison of libraries prepared from genomic DNA vs intact cells We hypothesized that tagmentation could be applied directly to heat-treated yeast spheroplasts to simplify whole-genome sequencing library preparation by skipping genomic DNA isolation
Summary
Whole-genome sequencing is a powerful tool in genomics research by providing an unbiased and comprehensive view of the genetic alterations present in a cell. The most rapid and scalable library preparation methods use a hyperactive variant of the Tn5 transposase that fragments double-stranded DNA and ligates synthetic oligonucleotide adapters required for Illumina sequencing in a 5-min reaction (Adey et al 2010) (Illumina). In addition to a hyperactive Tn5 enzyme variant carrying the previously reported missense mutations E54K (Zhou and Reznikoff 1997; Zhou et al 1998) and L372P (Weinreich et al 1994), which increase the DNAbinding efficiency and reduce inhibitory effects on Tn5 activity, respectively, we introduced a second Tn5 construct carrying an additional amino acid substitution (R27S) in the DNA-binding domain (Hennig et al 2017), which allows adjusting the fragment size distribution based on enzyme concentration during tagmentation
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