Hi Chris,

It may be that your reads no longer meet the default threshold parameters for Bowtie. To start, running a tool such as FastQC will give you some information about the length, quality distribution, and other metrics of your data. Doing this before and after trimming would likely be helpful, and perhaps guide you to the optimal trim options.

Next, open the "Bowtie settings to use: Full parameter list" (default is "Commonly used"). Comparing the values to their meaning in the documentation and to your data will likely pin-point the problem. For example, if you have trimmed most of your data down to 25 bases, but have a "Seed length (-l):" of 28 (bases), then most of your data will not align.

Short documentation is on the tool form to be convenient, for example:

-l INT Seed length. The number of bases on the high-quality end of the read to which the -n ceiling applies. Must be at least 5. [28]

-n INT Mismatch seed. Maximum number of mismatches permitted in the seed (defined with seed length option). Can be 0, 1, 2, or 3. [2]

Best wishes for your project,

Jen Galaxy team

On 3/1/12 8:57 AM, Buxton Chris (NORTH BRISTOL NHS TRUST) wrote:

Hi, I hoped that someone could shed some light onto this . I am attempting to map a set of 2x 150 illumina PE data from a DNA resequencing project. The run had an issue where the quality of the last 50 or so reads of the second run tail off quite considerably. I thought to trim the second read such that the poorer sequence bases are stripped form the end of the read. I attempted to do this using the FASTQ quality trimmer then mapping both reads using bowtie. When I did this however, I went from an alignment of 34% passing filter reads aligned not trimmed (which is not good to start off with), to 0.18%. trim command was set as defaults: Any ideas what I could be doing wrong?

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