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Pipeline publication

[…] reciprocal BLAST heuristic implemented in the program Proteinortho v.4.25 (settings: -e = 1e-10, -p = blastp, -id = 25, -cov = 0.5, -conn = 0.1, -m = 0.95) . Orthologs were required to be in single copy and full-length, i.e. not split on two or more contigs., Data of the same library but from different sequencing lanes were merged. The data were then searched for read-pairs containing the library adapter. Adapter sequences were identified in 45 to 61% of the read-pairs, indicating sequencing of DNA fragments <200 nt. Before the data were mapped, adapter-containing read-pairs were identified and the overlap of left and right reads was used to join the mates into one sequence. The program SeqPrep (downloaded May 21, 2012; https://github.com/jstjohn/SeqPrep) was used to join these reads (non-default parameter: −6). Joined reads were then mapped as single-end reads to the genomes. Moreover, read-pairs without the adapter were mapped as paired-end reads, since these read-pairs resulted from sequencing of DNA fragments >200 nt. All reads were aligned with bowtie v.2.0.0-beta6 . Reads reporting more than one match were kept pseudo-randomly using bowtie's default options., Transcript levels were calculated from the mapped sequences as fragments per kilobase per million fragments mapped (FPKM), using cufflinks v.2.0.0 . The setting ‘–library-type fr-secondstrand’ was used. The threshold for transcription was set to 0.5 FPKM; i.e., genes with FPKM≤0.5 were not regarded expressed., Total RNA from four 10 ml cultures of G. intestinalis WB trophozoites was prepared using the TRIzol reagent as specified by the manufacturer (Ambion, Life Technologies). RNA samples were pooled and treated with DNAse-I (Fermentas). The quality and quantity of the RNA preparation were assessed with TBE-agarose gel and Nanodrop. Polyadenylated cDNA was prepared using the Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit using 1.5 to 2.0 µg of DNAse-I treated total RNA per reaction. Minus template […]

Pipeline specifications

Software tools SeqPrep, Bowtie, Cufflinks