Computational protocol: Volatility of Mutator Phenotypes at Single Cell Resolution

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Protocol publication

[…] For sequencing, the entire daughter colony was used to inoculate overnight YPD cultures. Glycerol stocks of each daughter culture were archived and genomic DNA was purified with a ZR Fungal/bacterial purification kit (Zymo Research). The purified DNA was simultaneously fragmented and ligated to Illumina DNA adapters using the Nextera V2 Kit (Illumina), post-indexed by PCR (primer sequences available upon request), and sequenced using 101 bp, paired-end reads on an Illumina 2500 platform. Once all members of a lineage had been sequenced, we used the Burrows-Wheeler Aligner (v0.6.2) [] to align the reads against a copy of the S288C S. cerevisiae genome (Assembly R64-1-1) in which low complexity and highly repetitive sequences have been removed (<0.5% of the genome) with RepeatMasker. After the initial alignment, unmapped and ambiguously mapped reads were filtered out. PCR duplicates were evaluated using the MarkDuplicates option in the Picard suite of programs (http://picard.sourceforge.net). To further reduce false variant calls, the Genome Analysis Tool Kit (GATK) suite of programs was used for local realignment and base quality score recalibration []. We used VarScan2 [] for variant calling with a minimum read depth of 15, a minimum variant frequency of 0.8, and a quality score of 15. We then filtered the resulting variants to remove strain-specific single nucleotide polymorphisms segregating in our genetic background using a database of putative SNPs segregating in the BY4743 strain background that we previously compiled []. We normalized coverage to ensure that we only scored mutations in sequences shared by all members of a lineage. Finally, to determine maternal and daughter specific variants, we compared the mutations found in the last daughter to those found in all preceding daughters. Variants found in the last daughter and shared by one or more of the preceding daughters were designated maternally fixed mutations (See ). Those not found in the maternal lineage were designated daughter-specific mutations and were not evaluated further for this study. Called mutations were then visually confirmed using the Integrated Genome Browser. […]

Pipeline specifications

Software tools BWA, RepeatMasker, Picard, GATK, VarScan
Application Genome data visualization
Organisms Saccharomyces cerevisiae
Diseases Neoplasms