Computational protocol: Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species

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Protocol publication

[…] Now the whole sequences for eight teleost species, including zebrafish, fugu (Takifugu rubripes), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), cod (Gadus morhua), coelacanth (Latimeria chalumnae), tetraodon (Tetraodon nigroviridis) and tilapia (Oreochromis niloticus), are available in the Ensembl database. We downloaded all protein sequences of the eight species from Ensembl, and then conducted a comparative transcriptomic analysis between crucian carp and these fish species using tBLASTx at E-value <1e−10. [...] The software MISA (http://pgrc.ipk-gatersleben.de/misa/) was employed to discover microsatellite sequences from the unigene sequences. Five types of microsatellites were identified with criteria of di- to hexa-nucleotides motifs, and the minimum repeat unit was defined as 6 for di-, and 5 repeats for tri-, tetra-, penta- and hexa-nucleotides. The sequences composed of two or more repeat units with motifs separated by >100 bp were considered to be two or more microsatellites. Only microsatellite sequences with flanking sequences of ≥50 bp on both sides were collected for future primer designing. We used QualitySNP (http://www.bioinformatics.nl/tools/snpweb/) to identify potential SNPs from isotigs containing at least 10 reads. Only those SNPs with a minor allelic frequency no less than 20% were identified. The indels were not included in SNP analysis. […]

Pipeline specifications

Software tools TBLASTX, MISA
Applications WGS analysis, Nucleotide sequence alignment
Organisms Carassius carassius, Carassius auratus