New with 454

[aicb]

Hi everybody!

I'm going to start working in a project with 454 and I am really new with it. I have never worked with NGS before, I've been to a couple of seminars about Ilumina, but I have no clue about 454 (I know the pyrosequencing basics, but I mean formats, data analysis and these stuff).
- Which kind of data does it produces (files, format, quality formats... etc)?
- Is the software included useful to analyze resequencing data?
- How does it work with RNA...

Could you recommend me any paper or web where I can read more about it? I am especially interested in the data analysis (which I guess is the most difficult part).

THANK YOU VERY MUCH! I really appreciate any help!

[drio]

1. Typically sff that can be easily converted to fastq for secondary analysis processing.

2. Roche provides a mapper, but you can also compute the alignments using open source tools. People speaks well about bwa-sw. Mosaik should perform very well too. Anyone?

About the documentation, start with this paper, it has links to the details of the chemistry in the references.

[aicb]

Drio, thanks a lot for the info!
Would you recommend to use the Roche mapper? I have been reading that it is not the best one... but I have no experience with other mappers and I guess it'll be easy to use the one that came with the machine...
Thanks again

[nickloman]

The Roche mapper is pretty good, certainly one of the best for 454 data. A good 454 mapper will understand the specific 454 error model (resulting in indels and homopolymer miscalls). That's why using short-read mappers which are designed with Illumina data in mind don't very well. You might also want to try LASTZ. Heng Li recommends SSAHA2 although I have little experience with it.

[aicb]

Thanks Nickloman! I'll look for those programs.
Another question, I would also like to work with transcriptome, does it work the same for RNA? Anyone knows which is the limit of detection? Any specific program? Do you know of any good review or document that I can read about RNA with 454.
Thanks

[nickloman]

There are plenty of RNA-Seq publications using 454, I'd suggest browsing the papers here for methods:

http://454.com/publications-and-resources/index.asp

You can filter them by "transcriptome sequencing"

[aicb]

Another question... is it possible to sequence a sample in 454 with RNA from two different organism? like clinical samples or animal models samples?
THX

[nickloman]

I'm not sure exactly what you mean by your question. I suspect the answer is "yes" but perhaps you could be more specific.

[aicb]

I want to extract RNA from a clinical sample (like a biopsy from a lung) so it should have the human RNA and the bug RNA, and sequence it. It'll have a mix of both RNAs, I guess that after getting the reads I could mapp it with the two genomes (both are sequenced) so I'll know which read belongs to each organism... Am I right? is that possible? can I quantify each of the RNAs latter on? am still lost with these, sorry for my ignorance... I really appreciate your help!

[rwenang]

this might be helpful...

[westerman]

rwenang's paper looks useful. I point out that people have been for many years doing sequencing with mixed environmental samples and getting useful results. i.e., sequence a vial of lake water and see what pops up. Your environment is only two organisms (although I suspect that you will pick up more) and thus should be feasible.

[aicb]

Thanks! that was really helpful.