Additional file 1:

Normalized sample did not perform equally in pilot and full sequencing runs. (A) For the normalized sample, the read lengths of the full plate sequencing runs (white) were shorter than those obtained by the 1/8 plate run (grey). (B) The read length distribution of the non-normalized sample was comparable for both 1/8 plate (grey) and full plate (white) sequencing runs.

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Additional file 2:

Distribution of average coverage (reads/bp) within contigs in the O. fasciatus transcriptome. The coverage within contigs is calculated by dividing the total number of base pairs contained in the reads used to construct a contig by the length of that contig. Note that Newbler v2.3 discards those contigs <100 bp.

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Additional file 3:

RT-PCR validation of bioinformatically predicted multiple isoforms. (A) Schematic of experimental design. Ten isogroups were randomly selected, each containing exactly two isotigs that differed by the presence/absence of a single contig. PCR primers were designed to flank the differing region. (B) Band sizes predicted by Newbler v2.3 for ten randomly selected isogroups containing exactly two isotigs. (C) Agarose gel following RT-PCR using primers against the sequences described in (B). Ladder sizes are given in base pairs on the left. Blue arrowheads: bands of the sizes predicted by Newbler v2.3; red arrowheads: bands not predicted by Newbler v2.3.

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Additional file 4:

Identity of taxa with top BLAST hits. "Isotigs" refers only to the longest isotig of each isogroup; "Singletons" refers to the Newbler-generated singletons after secondary CAP3 assembly. The category "other" is the summation of all those species obtaining very low numbers of BLAST hits.

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Additional file 5:

O. fasciatus assembly isotigs have ortholog hit ratios similar to predictions from fully genome-sequenced databases. When isotigs from the O. fasciatus transcriptome are BLASTed against the RefSeq protein database, ortholog hit ratios show a similar profile to those obtained when the complete Acyrthosiphon pisum gene prediction set (downloaded from http://www.aphidbase.com/aphidbase/downloads/ webcite) is BLASTed against the predicted gene set of Drosophila melanogaster (r5.28 downloaded from ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/ webcite) with an e-value cut-off of 1e-10.

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Additional file 6:

GO terms enriched in Normalized (N) and Non-Normalized (NN) cDNA samples. N (assembly generated from full plate of normalized cDNA) and NN (assembly generated from an equalized number of base pairs of non-normalized cDNA) reads were BLASTed against the full transcriptome assembly, and the results were used to generate "test" and "reference" sets for a Fisher's Exact Test. FDR: false discovery rate.

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Additional file 7:

Comparison of de novo transcriptome assemblies produced by Newbler v2.3 and Newbler v2.5. Number of BLASTx hits reflects a search against RefSeq Protein database with an e-value cut-off value of 1e-10.

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