Computational protocol: Forward Genetic Screen in Caenorhabditis elegans Suggests F57A10.2 and acp-4 As Suppressors of C9ORF72 Related Phenotypes

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Protocol publication

[…] Synchronized L1 animals were seeded on 10 NMG plates (8.5 cm diameter), grown until they were gravid adults, and then washed off with M9 buffer and harvested in several 50 ml Conical tubes. After rinsing with M9 2–3 times, the animals were left on a NUTATOR mixer for 2 h to eliminate food in the gut, then were washed again with M9 2–3 times. The supernatant was removed after centrifugation at 1500 g for 3 min. A total 500 μl worm pellet was achieved by such method. For DNA extraction, we followed the protocol of Gentra Puregene Kit (Qiagen). The concentration of the final DNA preparation was determined by the Qubit assay (Invitrogen) at the Bauer Core Facility, Harvard University. 1 μg DNA was used for further sequencing library construction and next generation sequencing at BGI Americas (Cambridge MA USA). In brief, the sequencing libraries were made per the TruSeq®DNA PCR-free sample preparation kit (Illumina), and next generation sequencing was performed on an Illumina HiSeq seq platform. The sequencing results were analyzed both at BGI Americas Inc. and at the High-performance Computation Facility of the Partners Healthcare System. In brief, the sequencing reads were first mapped to the reference genome, such as WS220, with Bowtie-0.12.7, the variants were called with Samtools, and they were visualized by the Integrative Genomics Viewer developed by the Broad Institute. Nonsense mutations producing nonsense-mediated mRNA decay (NMD) and canonical splicing site mutations were noticed and confirmed by Sanger sequencing. […]

Pipeline specifications

Software tools Bowtie, SAMtools, IGV
Application WGS analysis