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Pipeline publication

[…] b'mental stages, i.e. pre-infective (n\xe2\x80\x89=\xe2\x80\x897), infective (n\xe2\x80\x89=\xe2\x80\x897, > 50\xc2\xa0mg) and post-reproduction adults (n\xe2\x80\x89=\xe2\x80\x893, > 350\xc2\xa0mg) in a simulated bird gut. Worms were extracted from a population of lab-raised and artificially infected sticklebacks at the University of Leicester, England (UK). Details of the method and complete primer sequences are available in Additional file ., To eliminate potential fish cDNA contamination that could have been introduced in the transcriptome due to host tissues inadvertently left on the parasite\xe2\x80\x99s integument during the dissection, three different bioinformatics controls were used. First, cleaned reads were mapped to the raw genome of Schistocephalus solidus using BWA-SW [] with default parameters. Default parameters for the program BWA are designed to offer the best possible balance between performance and accuracy. The program also automatically adjusts parameters according to read length and error rates []. BWA default parameters are thus sufficiently efficient to achieve the goal of discarding low quality reads that do not match the reference genome. Reads that did not map on the genome were discarded from the assembly. The second control was performed at the end of the assembly. Final contigs obtained with both assembly methods (RAY and MIRA) were blasted against the raw genome of S. solidus and sequences returning no hits were discarded. A third control was finally used to confirm that the mimicry candidates found with pipeline B (Figure\xc2\xa0) were tapeworm proteins and not cDNA contamination from the host. Candidate proteins were blasted against the proteomes of the host and the parasite (blastp searches) and cDNA sequences corresponding to these proteins were also respectively blasted against the genomes of the host and the parasite (blastn searches), which allowed us to assess if these sequences (at the levels of nucleic acids and amino acids) were more similar to the tapeworm or the fish proteome. When the e-value, the raw bit score and the length of alignment were systematically greater when blasted against the parasite as compared to the host, candidates were considered as true parasite sequences and not contamination. We also performed a fourth control in the laboratory to determine whether the four candidate mimicry genes do originate from Schistocephalus solidus and not from threespine stickleback DNA contamination. To do so, we conducted a PCR validation experiment using DNA from three pools of coracidia, the fre' […]

Pipeline specifications

Software tools BWA, BLASTP, BLASTN