PeakNarrow ATAC-Seq analysis
2
2
Entering edit mode
4.1 years ago
Spacebio ▴ 200

Hello biostars,

I am new to this type of analysis. I have IDR.NarrowPeak data and I would like to normalise it to FPKM using R. I read that the packages limma and DESeq are helpful but I still have problems creating the count matrix. Is there a step I am missing?

Any help/suggestion will be appreciated.

atac-seq narrowpeak • 1.4k views
ADD COMMENT
1
Entering edit mode
4.1 years ago
igor 12k

You should try DiffBind, which uses DESeq and edgeR in the background and automates this process: https://bioconductor.org/packages/release/bioc/html/DiffBind.html

ADD COMMENT
1
Entering edit mode
2.0 years ago
ATpoint 54k

The peak file is the template for the count matrix. You can e.g. use featureCounts to get the counts per peak and sample directly from the BAM files:

## 1. Transform narrowPeak to the featureCount SAF format:
awk 'OFS="\t" {print $1"_"$2+1"_"$3, $1, $2+1, $3, "+"}' in.narrowPeak > out.saf

## 2. Run fC:
featureCounts -a out.saf -F SAF -o countmatrix.txt *.bam

This countmatrix you can normalize with e.g. DESeq2 or edgeR or transformations such as vst and rlog. Stay away from naive methods such as R/FPKM. They are not suitable for inter-sample comparisons.

ADD COMMENT

Login before adding your answer.

Traffic: 2430 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6