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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