For constructing a molecular phylogenetic tree for FAS and FADD proteins, we used Fas and FADD sequences manually predicted from the chicken, zebrafish and stickleback genomes together with published catfish (Ictalurus punctatus), frog, human and mouse Fas and FADD sequences [ 9, 10, 35- 40] [see Additional file 7].
dSequences manually predicted de novo (see Methods).
Currently, such information is manually curated from the literature, obtained from high-throughput microscopy-based screens and predicted from primary sequence.
Coding sequences of these Drosophila species were predicted from genomic regions containing BLAST matches using the programs Genscan [18] and SNAP [19], and manually adjusted based on ortholog alignments.
By comparing the chemokine sequences predicted from the zebrafish draft sequence with those of isolated cDNAs and ESTs, the sequences have been manually curated to ascertain a higher level of completeness and quality.
We manually curated all nine cDNAs and showed that they were all reliable based on our validation criteria (Table 2), which included the removal of the uORFs predicted from ribosomal rRNA genes (data not shown).
FT was performed following cleavage of heterocyclized peptide by chymotrypsin or PatA protease, using a LTQ FT Ultra Hybrid Mass Spectrometer (Thermo Scientific) and analyzed both manually using predicted masses from Monoisotopic calculator and using Mascot from Matrix Science.
The full list of the top 100 prediction results can be obtained from the Additional file 2. Furthermore, we manually checked the top 10 predicted associations from the latest online versions of HMDD [ 17], miR2Disease [ 18] and dbDEMC [ 19] databases.
For manually annotated gene families, the predicted proteomes from individual fungal genome sequences were screened using BLASTP (Altschul et al. 1997) in an iterative process, using reference proteins that covered the diversity of the respective gene family (supplementary file S1, Supplementary Material online).
Proteins derived from orthologous lepidopteran genes that were not identified within the initial Lep1 screen (see 2.1), but present within the blastx output were compared manually to predict instances of copy number variation within or between species.
Since some protein sequences are 5′ truncated or predicted starting from an upstream point, we manually checked the prediction of top expression of transcripts containing signal peptides.
