Computational protocol: Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster

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Protocol publication

[…] Library construction for whole-genome sequencing was performed according to the manufacturer’s protocol (TruSeq DNA Sample Prep Kit v1, Illumina, San Diego, CA). Whole-genome resequencing was performed on Illumina HiSequation 2000 platform with paired-end 2 × 100 reads (Illumina). Adapter or low quality bases were trimmed from sequence reads. Then the cleared sequences were aligned to the reference genome dm3 using the BWA (). The Mark Duplicates program from Picard ( was used to remove duplicate reads. Local realignment, the recalibration of base quality values, and the adjustment of per-base alignment qualities were performed by components from the GATK pipeline (; ). The Unified Genotyper of GATK was applied to call the sequence differences or indels. Due to the fairly aggressive manner of the Unified Genotyper in making either SNP or indel calls, the raw call sets was filtered to reduce false-positive results based on call quality, depth, strand bias, etc., according to GATK best practice (version 4).Three mutant strains were exceptional in showing much higher frequencies of sequence differences. In each case there was a single large hypervariable region on chromosome 3R, within which apparent mutations occurred at much higher rate than normal (one mutation per 323 bp). These hypervariable regions were distinct but each partially overlapped another. This suggests that these three mutant chromosomes each contained sequences derived from a common progenitor that differs substantially from both the FRT82B chromosome and the reference genome sequence. We are unsure when these sequences entered our FRT82B strain, but sequences from these three anomalous regions have been excluded from our sequence analysis results. [...] Primers of 20–22 bp length and annealing temperature of 57–63° were designed for 500–700 bp PCR products. NCBI/Primer-BLAST was used to optimize primer design and minimize off-target sites. QIAquick PCR Purification Kit (Qiagen) was used to purify PCR products for Sanger sequencing. […]

Pipeline specifications

Software tools BWA, Picard, GATK, Primer-BLAST
Applications WGS analysis, qPCR
Organisms Drosophila melanogaster