Computational protocol: Avoidance of APOBEC3B-induced mutation by error-free lesion bypass

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Protocol publication

[…] Yeast transformed with A3B-expressing plasmid were plated on YPDA supplemented with hygromycin (300 μg/ml) at a density of ∼200 cells per plate and grown for three days at 30°C to accumulate mutations within independent colonies, after which they were replica plated to SC-arginine media supplemented with 0.006% canavanine and grown for an additional three days to select for CanR yeast. Alternatively, following a 6 h incubation at 37°C, cultures of cdc13-1 yeast transformed with A3B were directly plated to SC-arginine media supplemented with 0.006% canavanine and allowed to grow four days at 23°C to obtain CanR isolates. Independent CanR papillae were then selected and further clonally isolated through single colony streaking. Genomic DNA was isolated by bead agitation in a LiAc/SDS/TE buffer and purified over OMEGA HiBind DNA mini columns (Omega Bio-tek, Inc., Norcross, GA, USA). The CAN1 gene in isolates from wild-type, ubc13Δ, mph1Δ, ung1Δ, ubc13Δ mph1Δ, ubc13Δ ung1Δ and mph1Δ ung1Δ genetic backgrounds were PCR amplified using primers with unique identification barcodes listed in . These amplicons were pooled and sequenced by SMRT sequencing on a PacBio RSII as in (). We obtained a total of 6197 consensus sequence reads, each obtained by at least three complete passages of an individual DNA fragment. This equated to 2–63× coverage per barcoded amplicon. The sequencing results were sorted into individual isolates identified by their unique identification barcode combination. Consensus sequence reads were assembled to the reference CAN1 gene and mutations called in Geneious 8.1.8 (Biomatters Limited, Newark, NJ, USA). All called mutations were supported by multiple aligned circular consensus sequences (CCS) and occurred in greater than 50% of aligned reads per unique barcode set. The CAN1 gene from mms2Δ and apn1Δ apn2Δ yeast strains were PCR amplified using alternative primers listed in and Sanger sequenced (Genscript Biotech Corporation, Piscataway, NJ, USA) as described in (). Statistical significance for strand bias or nucleotide insertion preference was determined using a G-test of goodness-of-fit. Comparisons between mutation spectra of different genotypes were evaluated using a two-tailed Fisher's exact test in Graphpad Prism5 (Graphpad, La Jolla, CA, USA). The preferred nucleotide sequences mutated in CAN1 following A3B expression were generated with the mutated base +/− 1 nt using WebLogo (http://weblogo.berkeley.edu/logo.cgi) as described in (). All mutations identified through sequencing are listed in and summaries of the mutation spectra are provided in . […]

Pipeline specifications

Software tools Geneious, WebLogo
Application Genome data visualization
Diseases Deficiency Diseases, Neoplasms
Chemicals Cytidine, Deoxyuridine, Uracil