454 cDNA output files

[elenisp]

Hallo everyone,
I work at the University of Amsterdam and we have recently done a 454 Titanium run on normalized cDNA from tomato tissue. I have used the software from Roche- GS De Novo Assembler, to align the reads (non-paired) from the ssf files. I was wondering if anyone has used the software and can answer some questions about the output files:
1) I have noticed that (within an isogroup) sometimes the sequence of an isotig doesn't match with the contig sequences- so the contigs (and reads) all align but the corresponding isotig has a totally different sequence
2) Also I see in some isotigs that reads are being added (towards the 3') that don't have any overlapping sequence with the reads before

If someone has noticed these things with the data they are analyzing, I would appreciate any comments!
Thank you,
Eleni

[flxlex]

I use the cDNA option a lot for assembly, not for mapping though. We noticed some chimeric isotigs (we ave not yet quantified this). The problems you describe sound strange. I would contact Roche about this. The cDNA module is very new and who knows, there could be bugs...

[greatwanglei]

hello you two
I am in Beijing, China and going to use the GS De Novo Assembler to align the single end reads from a transcriptome project. Since you have used this software, would you like to give me some advice as to how to set the Parameters (especially minimum overlap length, minimum overlap identity, isotig and isogroup thredholds, isotig count and isogroup count thredholds).

Thank you!
greatwanglei

[flxlex]

I would perhaps only focus on the first two, I once got the advice from 454 to use more stringent mi and ml: -mi 95 -ml 90.

Good luck,

flxlex

[greatwanglei]

many thanks. will have a try.