Computational protocol: An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

Similar protocols

Protocol publication

[…] The sequencing reads from the targeted sequence capture experiments were aligned to the mouse genome (build 37, mm9) using the program bwa version 0.6.1 []. Datasets generated by Roche on the 454 platform were mapped using bwa bwasw [] while the remaining datasets, generated on the Illumina HiSeq or GAIIx platforms, were mapped using bwa aln, with default settings, followed by bwa sampe with the default settings. The resulting sam files were converted to bam files and coordinate-sorted using SAMtools version 0.1.17 [] and PCR duplicates were subsequently eliminated using the program Picard MarkDuplicates, version 1.48 [].For each sample, nucleotide variants were identified within the intervals for which linkage had previously been determined. This was achieved by creating a pileup file of the linked region using SAMtools mpileup, using the option -q 20, followed by variant calling using the program Varscan version v2.2.8 [] using the 'somatic' feature and the settings -min-coverage 15 and -min-var-freq 0.3. Varscan somatic calls sequence differences between a case and a control sample; as control the sequenced exome from a different ENU mutant was used. The case and control exomes were constructed using the same library preparation methods and sequenced in the same deep sequencing run, but had different linked intervals. All exomes were mapped and processed in parallel, using identical settings, to minimize post-sequencing artifacts. The output from Varscan was manually screened for likely ENU mutations, appearing as heterozygous SNPs in the mutant and wild type in the control. These SNPs were in turn validated using the Sanger method.The custom capture and sequencing of the MommeD34 linked region was carried out in-house without a matched wild-type control; instead, a merge of the three MommeD8 deep sequencing samples previously sequenced by Roche was used as control. A merge of these 454 datasets was used in order to achieve greater read depth across the region. SNP calling was subsequently carried out as described above. Exome sequencing datasets generated in this study are accessible via European Nucleotide Archive (ENA) under accession ERP003831. [...] Total RNA was extracted from various tissues using TRI reagent (Invitrogen). cDNA was synthesized from total RNA using SuperScriptIII reverse transcriptase (Invitrogen) or AMV reverse transcriptase (Roche) and random hexamer primers. Quantitative real-time PCR was performed with the Platinum SYBR Green qPCR Super Mix -UDG (Invitrogen) with primers designed to span exon/intron boundaries. All reactions were performed in triplicates and normalized to Hprt or Gapdh. PCRs were run on a Viia7 (Applied Biosystems, Mulgrave, VIC, Australia) or on a Corbett Research Rotor-Gene (Qiagen). Cryp-Skip [] was used for splice site prediction. cDNA from mutant alleles was sequenced using Sanger sequencing. Primer sequences are provided in Additional file . […]

Pipeline specifications

Software tools BWA, SAMtools, Picard, GATK, VarScan, CRYP-SKIP
Databases ENA
Applications WGS analysis, WES analysis
Organisms Mus musculus
Diseases Cardiovascular Abnormalities
Chemicals Zinc