Question: Bowtie with -a --best --strata does not seem to limit alignments to the best strata
1
gravatar for hosheaa
3 months ago by
hosheaa20
hosheaa20 wrote:

I am using bowtie for illumina on the NGS Galaxy public server.

This is the code for the run that I copied directly from the output SAM file:

"bowtie -q -p 6 -S -v 2 -n 2 -e 70 -l 28 --maxbts 800 -a --best --strata --phred33-quals /galaxy/data/hg19/hg19full/bowtie_index/hg19full /galaxy-repl/main/files/021/026/dataset_21026769.dat"

After I convert to BAM file and view in IGV Browser, I can see alignments with 2 mismatches, some with 1 mismatch and even some with 0 mismatches. See the sample image:

image from ivg

It doesn't look like -a --best --strata is working. Can someone help?

ADD COMMENTlink modified 3 months ago • written 3 months ago by hosheaa20
0
gravatar for Jennifer Hillman Jackson
3 months ago by
United States
Jennifer Hillman Jackson23k wrote:

Hello,

From the tool documentation:

--strata Best strata. When running in best mode, report alignments that fall into the best stratum if there are ones falling into more than one. [off]

With full details here: http://bowtie-bio.sourceforge.net/manual.shtml#strata

The strata filter will limit the hits reported, per query sequence, to the "best". There can be more than one "best hit" for some cases as described in the link above.

If the goal is to only report one alignment per query sequence, try the option Report up to n valid alignments per read (-k) set at 1. Please be aware that you may miss important hits using this method. For this job, this option was left at default (report all alignments that meet other set criteria).

There are other ways to filter the output, both during alignment and after (Galaxy has several BAM filter tools wrapped and available at the public server http://usegalaxy.org). You might want to review the choices for alignment and post-alignment filtering, test a few out, compare the results, and then determine the optimal settings/workflow for your project. See the examples of Bowtie (version 1) parameter combinations and the results produced by each in the manual link above to better understand how options work together.

All of this said, moving to the updated version of the tool, Bowtie2, is strongly recommended. Tools and tool wrappers change over time as bug fixes are made (to either) and new features/options are introduced or removed. The Bowtie2 manual can be found here: http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml. Please note this change related to the concept of grouping/scoring alignments by "stratum":

Bowtie 2 does away with Bowtie 1's notion of alignment "stratum", and its distinction between "Maq-like" and "end-to-end" modes. In Bowtie 2 all alignments lie along a continuous spectrum of alignment scores where the scoring scheme, similar to Needleman-Wunsch and Smith-Waterman.

For reference, Galaxy tutorials covering common analysis workflows can be found here: https://galaxyproject.org/learn/

Hopefully, this helps you decide how to proceed! Jen, Galaxy team

ADD COMMENTlink written 3 months ago by Jennifer Hillman Jackson23k
0
gravatar for hosheaa
3 months ago by
hosheaa20
hosheaa20 wrote:

Thanks Jennifer. I wanted to use bowtie instead of bowtie2 because the documentation states that bowtie is still better for reads averaging less than 50. And the average length of my reads are about 30 or less (miRNA analysis). So, based on your experience, would you still recommend bowtie2 in this case?

ADD COMMENTlink written 3 months ago by hosheaa20
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