F Bartusch at 12:46, 28 October 2026

2026-10-28T12:46:27Z

← Older revision		Revision as of 14:46, 28 October 2026
Line 1:		Line 1:
	{{Softwarepage\|bio/bismark}}		{{Softwarepage\|bio/bismark/3.1.0}}

	{\| width=600px class="wikitable"		{\| width=600px class="wikitable"
Line 6:		Line 6:
	\|-		\|-
	\| module load		\| module load
	\| bio/bismark		\| bio/bismark/3.1.0
	\|-		\|-
	\| License		\| License

F Bartusch: Created page with "{{Softwarepage|bio/bismark}} {| width=600px class="wikitable" |- ! Description !! Content |- | module load | bio/bismark |- | License | GPL-3.0 - see [https://github.com/FelixKrueger/Bismark/blob/master/license.txt] |- | Citing | [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27(11), 1571-1572 (2011)] |- | Links | Bismark Documenta..."

2026-10-28T12:45:48Z

Created page with "{{Softwarepage|bio/bismark}} {| width=600px class="wikitable" |- ! Description !! Content |- | module load | bio/bismark |- | License | GPL-3.0 - see [https://github.com/FelixKrueger/Bismark/blob/master/license.txt] |- | Citing | [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27(11), 1571-1572 (2011)] |- | Links | Bismark Documenta..."

New page

{{Softwarepage|bio/bismark}}

{| width=600px class="wikitable"
|-
! Description !! Content
|-
| module load
| bio/bismark
|-
| License
| GPL-3.0 - see [https://github.com/FelixKrueger/Bismark/blob/master/license.txt]
|-
| Citing
| [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27(11), 1571-1572 (2011)]
|-
| Links
| Bismark Documentation: [https://felixkrueger.github.io/Bismark/] <br>
Bismark Repository: [https://github.com/FelixKrueger/Bismark]
|}

= Description =

Bismark maps bisulfite-converted reads (WGBS, RRBS, EM-seq, ...) to a reference genome and calls cytosine methylation in CpG, CHG and CHH context.

The module <code>bio/bismark/3.1.0</code> is the '''Rust rewrite''' of Bismark. Upstream validates its default output as byte-identical to Perl Bismark v0.25.1. We checked this on BinAC 2 with the upstream test data (all outputs identical) and with 10 million human EM-seq read pairs (identical alignments). It is much faster than the Perl version, mostly in the methylation extraction:

{| class="wikitable"
! Step (10M read pairs, GRCh38, 16 cores) !! Perl 0.25.1 (<code>--multicore 8</code>) !! Rust 3.1.0 (<code>-p 8</code>)
|-
| Alignment || 785 s || 699 s
|-
| Deduplication || 190 s || 169 s
|-
| Methylation extraction (with cytosine report) || 3067 s || 293 s
|-
| '''Total''' || '''4042 s''' || '''1162 s'''
|}

All classic tool names are available: <code>bismark</code>, <code>bismark_genome_preparation</code>, <code>deduplicate_bismark</code>, <code>bismark_methylation_extractor</code>, <code>bismark2bedGraph</code>, <code>coverage2cytosine</code>, <code>bam2nuc</code>, <code>bismark2report</code>, <code>bismark2summary</code> and more. Your existing commands keep working, but see [[#Moving from Perl Bismark 0.25.1|below]] for the thread options.

The module loads <code>bio/bowtie2/2.5.5</code> automatically. HISAT2 (<code>--hisat2</code>) and minimap2 (<code>--minimap2</code>) are optional and must be loaded separately. samtools is not needed.

= Quick start =

<pre>
#!/bin/bash
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --cpus-per-task=32
#SBATCH --mem=32G
#SBATCH --time=04:00:00

module load bio/bismark/3.1.0

GENOME=/path/to/genome_folder # prepared with bismark_genome_preparation
cd "$TMPDIR" # fast local scratch of the job

bismark --genome "$GENOME" -p 15 \
-1 /path/to/sample_R1.fq.gz -2 /path/to/sample_R2.fq.gz
deduplicate_bismark -p --parallel 4 sample_R1_bismark_bt2_pe.bam
bismark_methylation_extractor -p --gzip --bedGraph \
sample_R1_bismark_bt2_pe.deduplicated.bam

mkdir -p /path/to/results
cp *.bam *.txt *.gz *.html /path/to/results/ 2>/dev/null
</pre>

For 50 million read pairs against the human genome this takes about one hour.

= Preparing the genome =

<pre>
module load bio/bismark/3.1.0
bismark_genome_preparation --parallel 16 /path/to/genome_folder/
</pre>

* '''Large genomes:''' Bowtie 2 builds a large index (<code>.bt2l</code>) automatically for genomes above 2 GiB (human, mouse and most mammals); you do not need <code>--large-index</code>. Giving it anyway does no harm. Bismark finds the large index automatically when aligning.
* '''Use one file extension for all FASTA files''' in the genome folder. Bismark reads only the first non-empty group of <code>.fa</code>, <code>.fa.gz</code>, <code>.fasta</code>, <code>.fasta.gz</code> and silently ignores the others. For example, if your genome is <code>genome.fasta.gz</code> and you add a spike-in as <code>lambda.fa.gz</code>, only the spike-in is indexed. Rename the files so they all end the same way.
* Check the result: <code>grep -c '^>' /path/to/genome_folder/Bisulfite_Genome/CT_conversion/genome_mfa.CT_conversion.fa</code> must show the number of sequences of your genome.
* Bismark builds two indexes (C to T and G to A converted genome); <code>--parallel N</code> builds both at the same time with N threads each. '''Request <code>--cpus-per-task=32 --mem=64G</code>''' for <code>--parallel 16</code>: each index build needs about 25 GiB, and both run at the same time. For the human genome this takes less than one hour.
* Prepare the index once and reuse it for all samples.

= Choosing threads and resources =

All recommendations below were measured on BinAC 2 standard nodes (AMD EPYC 7543) with human GRCh38 and EM-seq reads (2 x 151 bp). Other genomes and read lengths need different times and memory, but the thread recommendations apply in general.

== Recommended job size ==

Request '''<code>--cpus-per-task=32</code> and <code>--mem=32G</code>'''. On BinAC 2, Slurm counts hardware threads as CPUs, so this gives you 16 physical cores with 2 hardware threads each. In our tests this was the most efficient job size: with twice as many cores, the alignment of one sample was only 1.26x faster.

If you have many samples, run one job per sample with these settings rather than fewer jobs with more cores.

== Alignment: use <code>-p</code>, not <code>--multicore</code> ==

Bismark runs 2 Bowtie 2 processes (4 with <code>--non_directional</code>), each with <code>-p N</code> threads. Choose

: <code>-p</code> = cpus-per-task / 2 - 1

so <code>-p 15</code> for <code>--cpus-per-task=32</code>. The 2 remaining hardware threads are used by Bismark itself.

{| class="wikitable"
! Setting (10M read pairs, 16 cores) !! Time
|-
| <code>-p 8</code> || 720 s
|-
| <code>-p 15</code> || '''551 s'''
|-
| <code>-p 16</code> || 599 s
|}

* <code>--multicore</code> splits the input and starts a full Bismark with its own copy of the index per worker. It was clearly slower than <code>-p</code> with the same number of threads (559 s vs 374 s on a full node) and needs much more memory. Results are identical either way.
* Do not use more threads than you requested: oversubscribed jobs get slower, not faster.

== Deduplication ==

Use <code>--parallel 4</code>. For 10M read pairs this reduces the time from 169 s to 36 s. The output is identical.

== Methylation extraction ==

* <code>--multicore</code> does not make the extraction of BAM files faster. The extractor already uses about 4 cores on its own.
* '''Use <code>--cytosine_report</code> only if you need it.''' It writes every CpG of the genome, including uncovered ones, and takes most of the extraction time and memory:
{| class="wikitable"
! Extraction (10M read pairs) !! Time !! Memory
|-
| <code>--bedGraph</code> || 106 s || 0.8 GiB
|-
| <code>--bedGraph --cytosine_report</code> || 293 s || 3.4 GiB
|}
: Many downstream tools can read the coverage file (<code>*.bismark.cov.gz</code>) directly. If you need the cytosine report later, create it from the coverage file with <code>coverage2cytosine</code>.

== Time, memory and scratch space ==

Measured with <code>--cpus-per-task=32</code>, <code>-p 15</code>, <code>--parallel 4</code>, human GRCh38:

{| class="wikitable"
! Read pairs !! Alignment !! Deduplication !! Extraction !! Extraction with cytosine report !! Scratch space during alignment
|-
| 10 million || 9 min || 0.6 min || 1.6 min || 4.8 min || 10 GB
|-
| 25 million || 23 min || 1.4 min || 3.9 min || 7.2 min || 26 GB
|-
| 50 million || 45 min || 2.6 min || 7.6 min || 11 min || 47 GB
|}

Rules of thumb:
* '''Time:''' about 1 minute per million read pairs for alignment, deduplication and extraction together. In jobs that share a node with other jobs, plan about 15% more.
* '''Memory:''' about 12 GiB for aligning against the human genome, '''independent of the number of reads'''. Deduplication and extraction stay below 4 GiB up to 50M read pairs. A job with <code>--mem=16G</code> processed 50M read pairs successfully; <code>--mem=32G</code> leaves a safe margin.
* '''Scratch space:''' about 1 GB per million read pairs during the alignment (temporary converted read files), plus the input and output files. Work in the job's <code>$TMPDIR</code> (local disk) and copy the results back at the end. Alignment reads and writes a lot of data (50M pairs: about 180 GB read, 130 GB written); doing this on the parallel file system is slower and loads it for all users.

= Moving from Perl Bismark 0.25.1 =

* Results are the same as with Perl Bismark 0.25.1. The <code>@PG</code> line of the BAM header therefore still reports <code>VN:v0.25.1</code>.
* '''Replace <code>--multicore</code> by <code>-p</code>''' for the alignment, and add <code>--parallel 4</code> to <code>deduplicate_bismark</code>.
* If you ran Perl Bismark with <code>--multicore</code>, a tiny fraction of your methylation calls may differ from a new run (399 of 58.6 million CpG report lines in our test). Perl's <code>--multicore</code> writes the BAM in a different order, so deduplication keeps different but equivalent duplicates. The alignments themselves are identical.

= Support =

If you have questions or problems, contact hpcmaster [at] uni-tuebingen.de.

BinAC2/Software/Bismark - Revision history

F Bartusch at 12:46, 28 October 2026