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Coverage Required For Transcriptome Annotation

i am planing to use GS FLX + system for crow transcriptome sequencing . how many sample i can pooled to get minimum coverage of 5x or more than that. its necessary to focus minimum coverage for effective transcriptome analysis or any other option are available.

rna seq
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updated by Ketil Ketil • written by nayaganviji • nayaganviji

why crows? I cannot think in a good justification to sequence those.

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written by JC JC

Because they are there?

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written by Ketil Ketil

that's a really scientific justification

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written by JC JC

I think the most important issue is to produce scientifically sound data analysis. A botched job on an human tissue sample is worth nothing compared to good results obtained by studying an exotic nematode that only exists in Peru.

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written by Istvan Albert Istvan Albert

yes, exotic thinks are cool, but try to get funding for that ...

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written by JC JC

1 answer

Transcriptome coverage is very difficult to do properly, since RNA is present in very variable amounts, and it is difficult to get libraries where all transcripts are expressed. Normalization helps somewhat, but you may lose some genes entirely in the process, and in any case, it won't help you get transcripts that weren't present in the libraries.

So, no, I don't think you will be able to get 5x coverage for the complete transcriptome. The best you can do is to use a large variety of libraries, including early developmental stages and immunostimulated ones, and sequence as much as you can.

PS: Why not Illumina? It'll give you more sequence at lower cost.

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written by Ketil Ketil

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