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Taken as a whole, our validation demonstrates that the majority of assembled contigs represent genuine transcripts.
Our genome annotation pipeline proved to be a simple and accurate method to identify a large proportion of genuine transcripts.
We examined in detail the 63 cDNAs which by our criteria were genuine transcripts from 2-rep EhSINE1s.
To maximise data quality and the inclusion of sequences representing genuine transcripts, we used a three-step assembly strategy.
Therefore, the unambiguous identification of TSSs requires an efficient measure to distinguish the 5′-ends of such processed or degraded mRNA ends from those of genuine transcripts.
Although the remaining 1,734 reliable but not significant signatures were not considered in this analysis, prior analysis suggests that these signatures are likely to represent genuine transcripts expressed at very low levels [ 11].
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The length of the transcript was in full agreement with transcripts initiated at PhilD, as determined by primer extension [12], confirming that the 116-nt transcript was the genuine transcript from PhilD.
Earlier work showed that most singletons are genuine transcript sequences and therefore it is justified to retain and analyze them further [ 14].
Reverse transcriptase-PCR analysis failed to identify a genuine transcript encompassing both the Chlamydomonas rpoBa and rpoBb codingregions [ 9]; however, distinct transcripts were found to be specific to these ORFs [ 9].
In any case, tRNA sources would be from genuine gene transcripts and/or from pseudogene transcripts, because the highly conserved A58 was substituted with U58 in six cases and with C58 in one case among the nine 3'-half-tRNAs (Table S1).
However, in many cases multiple tags represent genuine alternative transcripts (e.g. alternatively spliced or polyadenylated forms).
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