Figure 3

Newbler 2.3 correctly identifies splicing isoforms of nanos. (A) Newbler v2.0 identified three separate contigs that map to an O. fasciatus nanos homologue that we had previously identified by degenerate PCR (Ewen-Campen & Extavour, unpublished). Newbler v2.0 failed to identify these contigs as belonging to the same transcript because of branching conflicts amongst the reads joining these contigs. BLASTX against the RefSeq protein database identified only contig 31035 as being a putative nanos homologue; the other two contigs lie outside the conserved Nanos domain and obtain no BLAST hits. (B) Newbler v2.3 predicted that the same three contigs identified by Newbler v2.0 belonged to two isotigs, or splicing isoforms. (C) RT-PCR with specific primers F and R shown in (B) resulted in two bands of the predicted sizes of the isotigs predicted by Newbler v2.3. (D) Sequencing the bands from (C) revealed that they were identical to the sequences of the predicted isotigs from (B).