Question

Hi, I recently performed an RNA immunoprecipitation followed by SOLiD sequencing (50 bp fragmented reads). I haven't received my first SOLiD sequencing results yet, but I was told I should have them soon. I've tried doing my own research on how to map, align, and plot my results but I don't have a concrete workflow as to how I will analyze my results yet. I have very little experience doing any programming and would prefer to use galaxy. There are labs on my campus I can go to to get my color space data mapped, but I would like to do things myself. Is there a way on galaxy (or another program) to convert my color space data to sequence, then map those reads to the yeast transcriptome and analyze it? Even if you can't answer my question directly I'd appreciate any tips from anyone who has worked with RNA-seq data already.

Thanks in advance

Answer 1

Personally I would advise that if you know someone who can partially perform the task you should have them do it, and ask them to explain and show it to you how they've done it.

The task at hand is complex. The solution always depends immensely on the particulars of the problem, moreover you will be facing myriads of frustrating limitations, errors and problems.

Learning directly from someone who has done it, establishing a personal rapport with them will allow you to ease into this problem domain. In fact when you are finished mapping your RNA - your are still likely to be far from being done - yet you might have expanded a lot of energy and excitement.

Answer 2

First of all, you should not convert to base sequence first and then map - you should do the mapping directly on the color-space reads. The short-read mapper will (typically) report the genome matches for you in base sequence format. There are several short read mappers / aligners that handle color space alignment: Bowtie, BFAST, BWA, SHRiMP, PerM and many others including ABI's own mapreads and Bioscope. You can get the mapping output in SAM format, a handy format which contains a lot of information about the alignments and which you can manipulate in Galaxy (via the NGS: SAM tools menu) to get the "pileup" of reads in certain regions and so on.

Edit: I just noticed that Galaxy now features Bowtie mapping for color space.

Answer 3

You can try BWA as well: http://maq.sourceforge.net/bwa-man.shtml