Evolution of the genomes of two nematodes in the ... - Ken Wolfe

(a) if it could only be aligned using T-COFFEE (Notredame et al., 2000) to < 25% of the lengths
of its top two matches in Caenorhabditis or in SwissProt 40.38 (Boeckmann et al., 2003);
(b) if it did not have any BLASTP hit in Caenorhabditis or SwissProt, of E-value < 10 −10 with
the SEG filter on (Wootton and Federhen, 1996), or < 10 −20 with SEG off; or
(c) if it had a within-species match, but no cross-species match, and was < 50 amino acids long.
This yielded the final (G3) gene sets for C. elegans and C. briggsae.
3.5.2 Finding C. briggsae-C. elegans Orthologues
We ran NCBI BLASTP (Altschul et al., 1997) with the C. briggsae protein set as the query database and
the C. elegans WS77 ∗ protein set as the target database, and vice versa. For C. elegans WS77 ∗ genes
that have alternative transcripts, we only took the longest splice variant.
We found orthologues in this way:
1. We found C. briggsae-C. elegans gene pairs that were each other’s top BLASTP hits. We required
the BLASTP hits to have an E-value of < 10 −10 with the SEG filter (Wootton and Federhen, 1996)
on, or < 10 −20 with SEG off. Furthermore, to avoid assigning paralogues to orthologue pairs, the
top hit had to have an E-value 10 5 times lower (more significant) than the next best hit.
2. We found additional orthologues by analysing conserved gene order. We found syntenic blocks by
looking for orthologues A (found in step 1) that were nearby to orthologues B (also found in step 1)
in both species. We identified C. briggsae-C. elegans gene pairs within the A-B syntenic block that
were each other’s top BLASTP hits within the A-B block (although not each other’s top BLASTP
hits within the genome). To avoid assigning paralogues to orthologue pairs, the top hit had to have
an E-value 10 5 times lower (more significant) than the next best hit in the A-B syntenic block.
3. Furthermore, we identified C. briggsae-C. elegans gene pairs that were each other’s top BLASTP
hits and that were within 100 kb of orthologues C (found in step 1) in both species.
3.5.3 Detecting Intron Gain and Loss in Orthologues
We used T-COFFEE (Notredame et al., 2000) to align all C. briggsae-C. elegans orthologue pairs. We
then searched the alignments for cases where exon i in species A aligned well to two adjacent exons j
and k in species B. To ensure that orthologous exons were matched properly, we required that exons i
and j, and exons i and k, had to consist of identical or conserved amino acids across at least 20% of the
shorter exon.
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