MACS3 is reporting that 100 paired reads are needed, even when single end data is used as input

[MingL196]

Description of the bug

I have some low quality data where the second paired read had very low quality. As such, I tried to run chipseq version 2.1.0 with single-ended data as input.

This results in an error with the MACS3 step. MACS3 is reporting that 100 paired reads are needed:

WARNING @ 09 Jul 2026 02:09:00: [86 MB] #2 MACS3 needs at least 100 paired peaks at + and - strand to build the model, but can only find 0! Please make your MFOLD range broader and try again. If MACS3 still can't build the model, we suggest to use --nomodel and --extsize 147 or other fixed number instead.

Command used and terminal output

nextflow run nf-core/chipseq --input samplesheet.csv --read_length 150 --outdir mouse_output_single_end2 --genome GRCm38 -profile singularity -r 2.1.0

Relevant files

No response

System information

No response

[JoseEspinosa]

Thanks for reporting @MingL196
MACS3's "paired peaks" message is unrelated to paired-end sequencing. It refers to an internal step where MACS builds its fragment-size shift model for single-end data, by looking for locations where a +-strand tag cluster sits near a --strand tag cluster ("paired peaks" in MACS's terminology). This step only runs for single-end input, which actually confirms the pipeline correctly detected and processed your data as single-end.
Finding 0 paired peaks means MACS3 couldn't build that model, consistent with what you described regarding the low quality of your data.
To try to make MACS3 work you can skip modeling step entirely with --nomodel --extsize <fragment_size> by overriding ext.args for MACS3_CALLPEAK in a custom config passed via -c:

process {
    withName: 'MACS3_CALLPEAK' {
        ext.args = [
            '--keep-dup all',
            params.narrow_peak          ? '' : "--broad --broad-cutoff ${params.broad_cutoff}",
            params.save_macs_pileup     ? '--bdg --SPMR' : '',
            params.macs_fdr             ? "--qvalue ${params.macs_fdr}" : '',
            params.macs_pvalue          ? "--pvalue ${params.macs_pvalue}" : '',
            params.aligner == 'chromap' ? '--format BAM' : '',
            '--nomodel',
            '--extsize 200'   // see note below on picking this value
        ].join(' ').trim()
    }
}

One important thing is that ext.args assignments for the same process replace rather than merge across config files, so this override has to re-declare all the other conditional args the pipeline normally sets (--keep-dup, broad/narrow, fdr/pvalue, the chromap format flag), you can't just append --nomodel --extsize on its own without losing the rest. I copied the current logic from modules.config so nothing gets silently dropped. For getting the current setup, you can easily inspect the command.sh resolved in the work directory of the process.

[MingL196]

The suggested config didn't work, with the error message:

"ERROR ~ Unknown config attribute process.withName:MACS3_CALLPEAK.params.narrow_peak -- check config file"

I was, however, was able to directly add
--nomodel --extsize 200
to
dev/modules/nf-core/macs3/callpeak/main.nf
and re-run the modified pipeline. This worked.

Thanks!