library compatibility

[Laruo]

Hi everyone!

does anybody now if a library constructed for Ion Torrent could be sequence in a 454 equipment directly? or if any additional step should be done?

have you used sometime IonTorrent for the quality control of a 454 library? similarly to the QC application of the Miseq for Hiseq.....

Thanks!!

[krobison]

No, each vendor has unfortunately chosen different adapter sequences.

If you amplified an Ion Torrent library using primers which had 454 adapter sequences 5' fused to Ion adapters 3', you could "convert" a library, but the beginning of your reads would be the Ion Torrent adapters and the PCR might skew the distribution of fragments and reduce complexity.

[Laruo]

Thank you krobison!

I was going to run a transcriptome library in Ion Torrent just for assess its quality before sending to sequencing in a 454 FLX, I have read about this application of Ion Torrent, unless it is true that it is not mentioned its utility for later sequencing in 454....

so... I'll have to think in something else

thanks again!

[krobison]

I'll confess a certain bias against 454 but I think you should ask yourself what the advantage of 454 is here over Ion or Illumina. For RNA-Seq applications, 454 has a bit longer length but many fewer reads in a run -- or more importantly, many, many fewer reads per dollar spent on sequencing. Depending on your objectives for RNA-Seq, in general more reads are more important than length. More reads means seeing rarer transcripts

Also, I realized later another issue here. 454 tolerates longer inserts than Ion Torrent. So if you have an Ion Torrent library, you won't get much longer reads on a 454 -- mostly you'd be just spending a lot more money.

[Laruo]

I understnasd the last you say...
maybe I could prepare a 454 transcriptome library but first run it in an Ion Torrent, with the modifications needed regarding adapters....I don't know, i have to think about it
Thank you anyway

[jdelton]

The "A" linker for 454 and Ion are the same (as far as the 454 rapid library linkers are concerned).

[ajthomas]

I'll confess a certain bias against 454 but I think you should ask yourself what the advantage of 454 is here over Ion or Illumina. For RNA-Seq applications, 454 has a bit longer length but many fewer reads in a run -- or more importantly, many, many fewer reads per dollar spent on sequencing. Depending on your objectives for RNA-Seq, in general more reads are more important than length. More reads means seeing rarer transcripts

Also, I realized later another issue here. 454 tolerates longer inserts than Ion Torrent. So if you have an Ion Torrent library, you won't get much longer reads on a 454 -- mostly you'd be just spending a lot more money.

I run a 454 and have used it exclusively up to now, but I have to agree here. I tried doing some RNA-Seq on the 454 and it just didn't work. Sure, I got data, and good data too, but I couldn't do any real expression analysis because there just wasn't enough. Not even close to enough. I'm now preparing libraries from those same samples to send off for sequencing on a HiSeq because my experience proved to me that the 454 just is not an appropriate platform for RNA-Seq.

If your goal is to construct full-length transcripts to find splice variants and such, it might be a good choice, but if you want to quantify expression at all, don't bother.