Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

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 limits 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 yeast cultures in less than three hours at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting 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.