Question: Bowtie2 alignment help for ATAC-seq
0
gravatar for mrs.whirly
12 months ago by
mrs.whirly0
mrs.whirly0 wrote:

Hello,

I am trying to align my raw sequencing data using bowtie2. This is for ATAC-seq analysis. What are the best commands settings to use on bowtie for that? I know I want paired-end alignments and local sequencing, but I am not sure what other options are good for ATAC-seq, such not including discordant pairs, limitations to concordant pairs (e.g. orientation, distance etc.), or anything else! Any advice is appreciated!

Thanks,

ADD COMMENTlink modified 25 days ago by ElCascador20 • written 12 months ago by mrs.whirly0
1

This guideline also add -k 10 but gives no reason. I guess this may relative to some very short fragments?

ADD REPLYlink written 25 days ago by MatthewP760
1

The author of this page you link (John Gaspar) developed a peak caller (Genrich) for ATAC-seq that can use multimappers, I guess therefore they allow for multimappers during alignment. I personally do not do that as it has not formally been shown that this is robust. I guess if you publish this you would need to robustly show that the way the peak caller uses the multimappers is indeed meaningful and does not create flawed results due to the inherent uncertainty of multimappers.

ADD REPLYlink written 25 days ago by ATpoint40k

How about leaving everything else as the default and then just taking a look at the results. As a rule of thumb, it's best to start with more-or-less default settings on tools and then tweak settings further only after having a look at the results.

ADD REPLYlink written 12 months ago by Devon Ryan97k

I second this. I have been trying to find a proper justification for using this parameter but I did not come to anything conclusive. They indeed use multimappers in their methods: https://github.com/jsh58/Genrich#multimap

ADD REPLYlink written 25 days ago by ElCascador20
2
gravatar for ATpoint
12 months ago by
ATpoint40k
Germany
ATpoint40k wrote:

Leaving everything at default is just fine expect that I find --very-sensitive and -X 2000 helpful. --very-sensitive simply triggers a more sensitive alignment mode and -X 2000 will flag reads with TLEN up to 2kb as concordant, still I doubt this will have a notable effect compared to the defaults which are always a good starting point.

ADD COMMENTlink modified 12 months ago • written 12 months ago by ATpoint40k
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