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I am in the process of assembling and annotating the genome of a non-model organism, using almost exclusively short read (paired-end Illumina) data. Throughput is one obvious benefit of these data (high coverage for reasonable cost, error rates notwithstanding), but another benefit of RNA-seq data in particular is that they can be used in multiple ways. For example, I am mapping the RNA-seq reads to the genome to estimate transcript abundances and do some differential expression analysis, but I have also assembled the RNA-seq reads de novo to use as evidence for genome annotation.

There are, however, limitations to using RNA-seq-derived transcripts for annotation. Full-length cDNAs would facilitate much more reliable annotation. However, I have no intuition for the relative cost, time commitment, or complexity of extracting and sequencing full-length cDNAs vs next-gen sequencing. Can anyone here comment on this?

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  • $\begingroup$ What kind of organism? Splicing makes using RNA more difficult. Is there a good existing reference genome for you organism (if lacking in annotation)? Do you have a specific goal, such as biofuels, drug production fighting pathogenic or parasitic organism, or do you have X amount of money and want to do the best basic research? The type and depth of sequencing that is most cost efficient will vary with your goals. $\endgroup$
    – Stuart R. Jefferys
    Nov 29, 2012 at 20:29
  • $\begingroup$ "What kind of organism?" It's eukaryotic, a social insect to be precise. "Is there a good existing reference genome for you organism?" As I said, this is a non-model species, so I am the one assembling the genome. I don't know what your criteria are for a "good" genome: no physical mapping, gap filling, etc has been done yet, but the assembled sequence is close to the expected genome size and preliminary annotations have identified about the number of genes we would expect (perhaps a little bit less). $\endgroup$
    – Daniel Standage
    Nov 29, 2012 at 21:25
  • $\begingroup$ "Do you have a specific goal?" The PI of this genome project is primarily interested in the intersection of evolution, genomics, and behavior, and uses several species of social insects as model organisms for these subjects. $\endgroup$
    – Daniel Standage
    Nov 29, 2012 at 21:27
  • $\begingroup$ IMO you should do a full length cDNA sequencing (sanger) only for the doubtful cases (Small contigs/ contigs mapping with gaps/ low depth) $\endgroup$
    – WYSIWYG
    May 30, 2015 at 17:22
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    $\begingroup$ I would comment on this instead of an answer but I don't have the reputation to comment yet. So RNAseq is actually done on cDNA. Specifically after selecting for mRNA before making a cDNA library by its poly A tail etc... It sounds like you are thinking that RNAseq is on actual RNA molecules? Or is your question more about long sequencing of full length cDNA from RNA instead of fragmented? That comes down to an increased error rate from sequencing longer sequences (of DNA). There shouldn't be any loss of de-novo information, transcript abundance, as well as isoform abundance from alternative s $\endgroup$
    – akaDrHouse
    Oct 19, 2015 at 3:56

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