quaqc: QUick ATAC-seq Quality Control

Also works with any unspliced DNA-seq experiment! Compatible with plant genomes!!

Installation

Requires gcc/clang and GNU Make, tested with macOS and Linux. Basic install:

sh git clone https://github.com/bjmt/quaqc # Or download latest release cd quaqc make release-full make test # Make sure quaqc produces the expected outputs (optional) make install # Copy quaqc + manual to /usr/local (optional, may require sudo)

See INSTALL for configuration options.

Alternatively for conda users, grab it from Bioconda:

```sh

Example environment setup:

conda create -y --name quaqc -c conda-forge -c bioconda quaqc ```

Quick start

To see a basic description of all available parameters, run ./quaqc -h. For more detailed help, see the manual page.

quaqc comes built-in with sensible defaults, meaning few parameters must be specified for regular usage. (The hardcoded defaults can be changed before compilation by editing quaqc.h.) To run quaqc using the test data:

sh ./quaqc -v --output-dir . --peaks test/peak.bed --tss test/tss.bed \ --target-list test/target.bed test/reads.bam cat ./reads.quaqc.txt

This command changes the location of the output QC report to the current directory, and uses the peak and TSS files to generate additional stats. Since this example BAM is just a subset of reads contained within a small region of the Arabidopsis genome, the target.bed file restricts quaqc to only consider that region of the genome (and thus adjust how it calculates the stats). See a copy of the output report here.

Note that quaqc can also save the final reads passing all filters to a new BAM file via the -S/--keep flag. (This will increase the runtime.)

quaqcr

For those who prefer to analyze their data with R, a companion package quaqcr is available. This package contains functions for both executing quaqc from within R as well as analyzing all of its outputs made available when outputting the results in JSON format. See the README for a detailed tutorial.

Help

Please open an issue on GitHub.

Citation

Please cite the following journal article if you find this software useful in your research:

Tremblay, B.J.M. and Qüesta, J.I. (2024). quaqc: efficient and quick ATAC-seq quality control and filtering. Bioinformatics 40, btae649. https://doi.org/10.1093/bioinformatics/btae649

Additional use cases

Creating insertion BED files for use by MACS2/3

A common method for calling ATAC-seq peaks is to provide the reads as a BED file in order to trick MACS2/3 into using individual reads during pileup instead of the fragment coordinates in paired-end data. Using the --bed option, this can be done using quaqc, generating identical output as bedtools bamtobed. For example:

```sh quaqc -0 --bed Sample.bam

macs3 callpeak [...] \ --treatment Sample.bed.gz --format BED \ --call-summits --keep-dup all \ --shift -75 --extsize 150 \ --nomodel --nolambda ```

However since we only care about the 5-prime ends of each read (i.e. the insertion sites) when calling peaks this way, we can also add the --bed-ins flag to tell quaqc to only output the 5-prime coordinates of the reads with no additional BED fields, saving a decent amount of disk space in a way which will not affect MACS2/3.

Creating bedGraph files centered around the 5-prime ends of reads

Typically read pileups of ATAC-seq data (such as bedGraph files) are made in such a way as to visualize the sites of transposition, which are the 5-prime ends of the reads. This means needing to resize each read before creating a bedGraph file. Both of these steps can be performed simultaneously using quaqc:

sh quaqc --bedGraph --bedGraph-qlen 150 Sample.bam

In this example, a bedGraph file is created where each read is resized to 150 bp, centered around the 5-prime end of each read. (To use the original read sizes without resizing, simply set --bedGraph-qlen to 0.) This functionality can also be used to visualize the exact insertions, adjusting for the +4/-5 transposition shift:

sh quaqc --bedGraph --bedGraph-tn5 --bedGraph-qlen 1 Sample.bam

The +4/-5 shift can also be adjusted to use custom values via --tn5-fwd and --tn5-rev.

Filtering a single-cell ATAC-seq BAM by cell type

Using a set of barcodes, quaqc can subset a BAM by making use of the --rg-list and --keep options. For example, using the common CB SAM flag typically used for storing barcodes:

sh quaqc -0 -A --rg-list barcodes.txt --rg-tag CB --keep --keep-ext .MyCellType.bam Sample.bam

The -0 flag prevents quaqc from creating a QC report, thus making the output BAM the only file created by quaqc. The -A flag can be used if this BAM has already been filtered, and no additional filtering is desired.

Fast iteration of the effects of different read quality thresholds

quaqc is fairly performant, but it can still require some patience for decently sized BAMs (e.g., about one minute for 80 million reads from Arabidopsis ATAC-seq). To substantially speed up quaqc to iterate through different filtering thresholds, make use of the -n/--target-names to restrict quaqc to only look at single chromosomes. This is often enough to get an idea of the quality of the data while getting a several fold speed up (e.g., restricting quaqc to the first chromosome of Arabidopsis takes just over 10 seconds for the previously mentioned example).

For example, to try out several MAPQ thresholds for an Arabidopsis ATAC-seq sample by only scanning chromosome 1 (which is just '1' for this version of TAIR10):

sh for i in 5 10 15 20 25 30 ; do quaqc -n 1 -q $i -i "MAPQ >= ${i}" -O .quaqc.mapq${i}.txt Sample.bam done

Now, six different MAPQ thresholds have been tested in the time it would take to process the entire BAM file once across all chromosomes. (The -i/--title flag is used to add a unique title to each run.)

Quick motif footprints

The TSS pileup functionality of quaqc can be used to instead generate motif footprints. Note that these are only useful for testing, as no correction of base composition is peformed to reduce Tn5 transposase insertion biases. The output must be saved in JSON format to recover the footprint data, which can then be plotted with an external program (such as quaqcr).

In this example code, the --footprint preset will adjust the TSS pileup to produce single base resolution data of Tn5 transposase insertion frequency. The --nfr preset is also used to only consider nucleosome-free reads.

sh quaqc --nfr --footprint --tss motif.bed --json sample_motif.json Sample.bam

The metrics

See this annotated report for a description of the metrics.

A note on additional metrics

The set of metrics output by quaqc are those that I personally have found useful. If you can think of additional important ones, please create an issue on GitHub and I would be happy to implement them if it seems feasible to do so.

Extra

A note on the default Tn5 shift values (+4/-5)

It is standard practice to adjust the start coordinates of ATAC-seq fragments to account for the ~9 bp footprint of the Tn5 transposase by adding 4 bp to the start and substracting 5 bp from the end (+4/-5). However, some articles such as this one instead suggest +4/-4 might better capture the true Tn5 offset and allow for better estimation of the sequence composition bias of transposition. To use this alternative offset in quaqc, simply add --tn5-rev 4.

Information on input BAMs

quaqc is meant to work with unfiltered BAMs produced by sequence alignment tools such as bowtie2. However, some processing steps are recommended and/or required. These include:

• samtools fixmate so that quaqc can compute accurate mate and fragment size metrics for PE experiments (optional, though not having proper read mate data may require using the --use-nomate flag to stop quaqc from filtering all PE reads)
• samtools sort if the reads are not already coordinate sorted (required)
• samtools markdup (or an equivalent command) so that quaqc can compute read duplication metrics (optional)
• samtools index to generate an accompanying index file (optional; quaqc will create one if missing)

My personal recommendation is to run all of these commands simultaneously during sequence alignment. For example:

sh [bowtie2/etc ... ] \ | samtools fixmate -m -u - - \ | samtools sort -u - \ | samtools markdup --write-index - file.bam

This requires a sequence aligner which can output its results to stdout, as well as being grouped by read name. For further information check out this article.

quaqc only works with indexed BAMs, though actually indexing them before using quaqc is optional. If no index can be found then quaqc will attempt to create the index.

CRAM

As far as I can tell CRAM files work fine as well, as long as the reference is available locally. See the notes in the installation instructions about compiling quaqc with libcurl if you need to work with CRAMs depending on remote references. The Bioconda version is dynamically linked to the full version of HTSlib, making it fully compatible with any type of CRAM.

Long reads

Some quick testing shows that quaqc seems to work with DNA-seq long read BAMs too. However if you try it out and see obvious errors in the output, please open an issue and I will do my best to fix the problem. Overall this is probably the wrong tool to use to run QC on long reads. Keep in mind quaqc will not properly handle supplementary alignments.

Spliced reads

I have not tested quaqc with BAMs containing spliced reads (e.g. RNA-seq), but I expect most of the statistics (such as alignment and fragment lengths) would be incorrectly calculated. Even the number of reads may be wrongly counted, since quaqc is not set up to properly handle supplementary alignments.