tons of questions regarding a cDNA project

[nicedad]

Hi everyone,

I'm new to this cmmunity yet very new to NGS technology. Thus I'm joining you guys.
We conducting a research on transcriptum of an insect and just got the cDNA dataset from a third part company. The project will be carried on 454 FLX Titanium and Illumina. All I know so far that one should use newbler, which already got installed.

My first question will be very primative and may be disturbing for you


Which file one should consider to take the sequences of the proteins hence the genes to be blasted into BLAST database to predict genes and expressed proteins? and how to take the sequences?

Frankly, I've got tons of question, thus I'm asking whether someone can advise, step by step please?

cheers,

[flxlex]

Assuming you will be using newbler for your assembly, with apologes for the shameless self-promotion: check out this blog post on the output of a newbler cDNA assembly:
http://contig.wordpress.com/2010/09/...-output-files/. You would want to use the isotig files.

[grassgirl]

Another newbie

I'm completely new to sequencing as well and am in the process of data analysis from a trial 454 Junior cDNA library run.

flxlex, I've been to your site several times and thank you for the information. It was very helpful in understanding what the input parameters, what the output is stating, what a contig vs. an isotig vs an isogroup.

I've gone through a first run on GS Assembler (the gui version) and now am looking for further direction on a couple of things:

How do I batch blast my sequences without making files of 50 sequences of them and manually uploading those files one at a time? I'm sure there are scripts out there for linux commands, but running things in command line is new to me as well (I've learned some basic commands to access my files, run GS Assembler, etc.).

For that matter, what should I be blasting? Contigs or isotigs?

Also, I've been told that I should simply do one assembly, such as running GS de novo Assembler with default values and accept that one run as the final output, but that I should try other values for the input parameters to see how that changes things, as well as running other algorithms. I'm at a bit of a loss about how I would change the input parameters and why and what other algorithms I should try. How do I know which ouput is good, etc?

Oh, and another thing: what do I do with the qual data? When I get an ouput of isotigs, how do I link it to the qual data for the contigs?

OK, I've loaded enough questions into one post.

[grassgirl]

Ooops, that should say 'I've been told that I SHOULDN'T simply do one assembly'.