Quick Start

Assume that you have a BAM file from aligner such as bwa mem named ATACseq.bam.

1. Sort the BAM file to get a ATACseq.sorted.bam file:

   samtools sort ATACseq.bam -o ATACseq.sorted.bam

2. Make index from the BAM file to get a ATACseq.sorted.bam.bai file:

   samtools index ATACseq.sorted.bam ATACseq.sorted.bam.bai

3. Make genome information (chromosome sizes) from the BAM file to get a genome.info file:

   samtools view -H ATACseq.sorted.bam| perl -ne 'if(/^@SQ.*?SN:(\w+)\s+LN:(\d+)/){print $1,"\t",$2,"\n"}' > genome.info

4. Run HMMRATAC on the sorted BAM ATACseq.sorted.bam, the BAM index file ATACseq.sorted.bam.bai, and the genome information file genome.info:

   java -jar HMMRATAC_V1.2.4_exe.jar -b ATACseq.sorted.bam -i ATACseq.sorted.bam.bai -g genome.info

5. Filter HMMRATAC output by the score, if desired. Score threshold will depend on dataset, score type and user preference. A threshold of 10 would be:

   awk -v OFS="\t" '$13>=10 {print}' NAME_peaks.gappedPeak > NAME.filteredPeaks.gappedPeak

   To filter the summit file by the same threshold:

   awk -v OFS="\t" '$5>=10 {print}' NAME_summits.bed > NAME.filteredSummits.bed

   NOTE: HMMRATAC will report all peaks that match the structure defined by the model, including weak peaks. Filtering by score can be used to retain stronger peaks. Lower score = higher sensitivity and lower precision, Higher score = lower sensitivity and higher precision.

Samtools can be downloaded here: http://www.htslib.org/download/

Be sure to run HMMRATAC using the executable file, found here: https://github.com/LiuLabUB/HMMRATAC/releases For details on HOW to run HMMRATAC, see HMMRATAC_Guide.md, which contains a thorough runthrough of all parameters, output files and input requirements and troubleshooting.

HMMRATAC requires paired-end data. Single-end data will not work. HMMRATAC is designed to process ATAC-seq data that hasn't undergone any size selection, either physical or in silico. This should be standard practice for any ATAC-seq analysis. Size selected data can be processed by HMMRATAC (see HMMRATAC_Guide.md on --trim option).

If you use HMMRATAC in your research, please cite the following paper:

• Evan D. Tarbell and Tao Liu, HMMRATAC: a Hidden Markov ModeleR for ATAC-seq, Nucleic Acids Res. 2019 Jun 14. doi: 10.1093/nar/gkz533