Paired-end sequencing problem

[moritza]

Hello everybody,

I'm a PhD student and I'm working with RNA-Seq data. My particular interest is paired-end sequencing by Illumina technology and therefore I would like to get a deeper insight into this topic.

At the moment I would like to understand, if a whole transcriptome can be sequenced completely with a theoretically infinite read coverage. It should be possible that also very small transcripts are sequenced as well. My focus isn't the assembly of reads, but the sequencing procedure itself. To more clearly illustrate what I mean I constructed a small example:

We are assuming, that a transcriptomic paired-end sequencing with the read length of 150 bp and mean insert size of 200 bp is accomplished. Furthermore we care the most about the reads encoding small proteins (10-200 aa). Thus there may occur transcripts with a length less than 300 bp. My question is now, will these short transcripts also be sequenced? Or should they rather be sequenced by single-end or another technique 454/Ion torrent additionally, in order obtain reads from the whole transcriptome?

[SNPsaurus]

If you size select your library, you will not sequence DNA fragments shorter than the size selected. In your example, the mean insert size is 200 bp, so you might get insert sizes of 300 bp and 100 bp at reduced levels.

Single-end or 454 would not necessarily solve the problem. For instance, you can perform single-end sequencing on fragments that are 500 bp long. It isn't the read length or the sequencing technology so much as working to ensure your library contains a wide range of insert sizes.