Question: ATAC-seq DE analysis
1
gravatar for collmerr
9 weeks ago by
collmerr10
collmerr10 wrote:

Hello all. I'm trying to do differential expression analysis using ATAC-seq data sets, but I'm having trouble getting HTseq-count or featureCounts to actually count the data. Any good protocols to accomplish this?

Thanks!

ADD COMMENTlink modified 9 weeks ago by trausch1.0k • written 9 weeks ago by collmerr10
1

HT-seq or featureCounts are not really suitable (tedious work to prepare an input format with bed regions) for ChIP/ATAC data. Use bamCoverage or multiBamSummary from deeptools. All you need is BAMs and a bed file.

ADD REPLYlink modified 9 weeks ago • written 9 weeks ago by venu5.5k

Disagreed. See my comment below. bedtools does not really support paired-end data, while featureCounts perfectly does. Preparing a featureCounts input file from BED is as easy as:

awk 'OFS="\t" {print $1"."$2+1"."$3, $1, $2+1, $3, "."}' peaks.narrowPeak > peaks.saf
ADD REPLYlink written 9 weeks ago by ATpoint6.5k
3
gravatar for Devon Ryan
9 weeks ago by
Devon Ryan82k
Freiburg, Germany
Devon Ryan82k wrote:

Differential accessibility, not differential expression.

We tend to use CSAW for this. The full pipeline is here, which is essentially:

  1. Fragments are filtered (alignmentSieve) so only those <=150 bases (i.e., likely from open chromatin) are present.
  2. MACS2 is run on that.
  3. CSAW is run in R and given the peaks from 2 and the unfiltered BAM files that were input into step 1.

Alternatively, you could use diffBind in the last step. As Venu pointed out, you can use multiBamSummary with a BED file to get your counts, but CSAW at least has functions that also do that.

ADD COMMENTlink written 9 weeks ago by Devon Ryan82k
1
gravatar for b.nota
9 weeks ago by
b.nota4.6k
Netherlands
b.nota4.6k wrote:

The way I approach this, is to first use MACS2 for peak calling using all BAM files together. This will generate a bed file with coordinates of all peaks. Then I use bedtools, something like this:

bedtools coverage -b ${bamFile} -a All_peaks.bed > CoverageFiles/${bamFile%.bam}.txt

And then merge all columns with the read counts from the files for edgeR.

ADD COMMENTlink modified 9 weeks ago • written 9 weeks ago by b.nota4.6k

The issue with bedtools is one can not input multiple bam files at once. With multiBamSummary from deeptools, you can input as many BAM files as possible, it will generate one counts table for all BAMs.

ADD REPLYlink written 9 weeks ago by venu5.5k
1

Bedtools can count alignments from multiple BAM files (see http://bedtools.readthedocs.io/en/latest/content/tools/multicov.html)

ADD REPLYlink written 9 weeks ago by James Ashmore2.5k

+1. I completely forgot about multicov function. Thanks for adding.

ADD REPLYlink written 9 weeks ago by venu5.5k

As ATAC-seq is typically paired-end, I recomennd featureCounts from the subread package, which can handle this (bedtools cannot as far as I know), and is multithreaded.

ADD REPLYlink written 9 weeks ago by ATpoint6.5k

It would be helpful then to show how you do it with featureCount, see OP's remark in the question: "but I'm having trouble getting HTseq-count or featureCounts to actually count the data".

ADD REPLYlink written 9 weeks ago by b.nota4.6k
1

Sorry, did not notice that sentence. There we go:

featureCounts cannot take bed or narrowPeak files directly, but wants a custom format called SAF:

## Make SAF file (+1 because SAF is 1-based, BED/narrowPeak is 0-based)
awk 'OFS="\t" {print $1"."$2+1"."$3, $1, $2+1, $3, "."}' peaks.narrowPeak > peaks.saf

## Then run fc:
featureCounts -a peaks.saf -F SAF -T <int> -p -o peaks_countMatrix.txt *.bam

-T = number of threads; -p indicates paired-end data

ADD REPLYlink written 9 weeks ago by ATpoint6.5k
0
gravatar for trausch
9 weeks ago by
trausch1.0k
Germany
trausch1.0k wrote:

Another ATAC-Seq pipeline is available here. As part of the pipeline a count matrix is generated and an Rscript template for differential peak calling is part of the pipeline (script is here).

ADD COMMENTlink written 9 weeks ago by trausch1.0k
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