Handling Multimapped Reads with --keep_multi_map Across Different Aligners I am currently utilizing the nf-core/chipseq pipeline (v2.0.0) for ChIP-seq analysis with an interest in transposable elements. I have enabled the --keep_multi_map option and am using various aligners (STAR, BWA, and Bowtie2). I am trying to understand the specific behavior of each aligner when this option is active: 1. STAR: How are multimapped reads handled when --keep_multi_map is true? Specifically, does STAR select the best alignment or report a random alignment in the event of ties? 2. BWA/Bowtie2: Could you detail how these aligners manage multimapped reads under the same condition? I have experience using STAR outside of the nf-core pipeline with specific parameters tailored for RNA-seq, which influence how multimapped reads are treated. --outFilterMultimapNmax 5000: This parameter sets the maximum number of multiple alignments allowed for a read. If a read maps in more than 5000 locations, it's considered too many and won't be included. --outMultimapperOrder Random: When there are multiple alignments for a read that are considered equally good, this parameter instructs STAR to randomly select one of these alignments to output. --winAnchorMultimapNmax 5000: This parameter is similar to --outFilterMultimapNmax but applies specifically to the number of maximum multiple alignments that are tried to be anchored within a window of the sequence. @Jose Espinosa-Carrasco

None of this tools arguments are set in the pipeline as the aligners are by default not set to multimap. You will need to create a

custom.config

and provide the desired tools arguments to the pipeline as you desire as explained here The

--keep_multi_map

parameter only controls that multimap reads are not filtered by samtools, see here

@Jose Espinosa-Carrasco Thanks for clarifying the settings for multimapped reads. I have set up a custom configuration to ensure STAR handles the reads as required.Here is my Execution Command: nextflow run nf-core/chipseq \ -r 2.0.0 \ --input path/samplesheet.csv \ --outdir path \ --genome GRCm38 \ --read_length 100 \ --aligner star \ --keep_multi_map true \ -profile singularity \ -c path/star_custom.config # Specify the path to your custom config Here is my star_custom.config // Custom configuration for STAR aligner in the nf-core/chipseq pipeline process { withName: 'STAR_ALIGN' { // Apply custom STAR aligner parameters ext.args = '--outFilterMultimapNmax 5000 --outMultimapperOrder Random' } } Does this setup look correct to you? And is this correct way pipeline will accept my custom configuration?

Hi, it is nice to format the code for making it more readable, as shown below, markdownhelp

Using some code formatting helps with readability and finding the issue. Here are some tips about how to format code snippets nicely in Slack: http://tiny.cc/mdcodeblocks http://tiny.cc/slacksnippets

Regarding what your post, keep in mind that your current custom config will overwrite the arguments passed to

STAR

and not append them to the current ones, see here, including

--outReadsUnmapped Fastx

So you will also need to provide all of the arguments you need in your custom config, e.g.:

process {
  withName: 'STAR_ALIGN' {
    // Apply custom STAR aligner parameters
    ext.args = '--outFilterMultimapNmax 5000 --outMultimapperOrder Random --outReadsUnmapped Fastx moreArgumentsHere'
  }
}

#Default pipeline

If you go to the work directory of he process you would be able to check the actual command nextflow had run by inspecting

.command.sh

, this way you will be able to check whether the arguments have been overwritten, if you don’t know how to do it share your

.nextflow.log

here and I can try to help you, from what I see it seems to me that the arguments should be correctly overwritten

@Jose Espinosa-Carrasco Do you mean the contents of the .command.sh like this. I dont see all my custom .contig parameters here. Or am I looking into wrong file

Yes, something 🐠 is going on, I think it should have worked this way, but it doesn’t seem to… I have been testing locally and using the whole name selector should work:

process {
    withName: 'NFCORE_CHIPSEQ:CHIPSEQ:ALIGN_STAR:STAR_ALIGN {
    // Custom STAR aligner parameters, including defaults and handling for multimapped reads
    ext.args = '--runMode alignReads --alignIntronMax 1 --alignEndsType EndToEnd --outSAMtype BAM Unsorted --readFilesCommand zcat --runRNGseed 0 --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 5000 --outMultimapperOrder Random --outReadsUnmapped Fastx'
    }
}

@Jose Espinosa-Carrasco i used more explicit path in the withName directive:'NFCORE_CHIPSEQCHIPSEQALIGN_STAR:STAR_ALIGN' in custom.contig ( file attached)

yes, I think so

@Jose Espinosa-Carrasco Thanks for your help