featureCounts parameter queries - minOverlap, fracOverlap, fracOverlapFeature
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Lucy ▴ 60
@lucy-17014
Last seen 9 weeks ago
United Kingdom

Hi,

I have a couple of questions regarding parameter choices for featureCounts. I have stranded 150 bp paired-end reads.

I have currently chosen the following settings. As far as I understand, this will count fragments rather than reads, will ignore chimeric fragments, and will count multimapping reads fractionally (primary alignments only; I have set my STAR mapping to output all alignments with the best score as primary alignments).

featureCounts
-p -B −−countReadPairs -C -F GTF --verbose -M --fraction --primary
-A chromosome_name_alias.txt
-T 4
-s 2
-g gene_id
-a Homo_sapiens.GRCh38.103.gtf
-o counts.txt
input.bam


I am interested in whether you would recommend to adjust the fracOverlap, fracOverlapFeature, minOverlap and nonOverlap parameters. By default, minOverlap is 1 - is this sufficient to confidently assign a read to a feature?

I don't fully understand the maxMOp parameter. Is anyone able to provide a simple explanation?

Many thanks for the suggestions.

Best wishes,

Lucy

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In addition to this, would you recommend to check the fragment length using -P and to adjust the min and max fragment length (-d and -D)? I assume you would set these based on expected fragment sizes from your library prep. protocol (in my case NEBNext Ultra II Directional).

What is the default behaviour is --largestOverlap is not set? I assume the fragment is just not assigned to any gene?

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Wei Shi ★ 3.5k
@wei-shi-2183
Last seen 4 days ago
Australia/Melbourne/Olivia Newton-John …

I wouldn't recommend changing the default values of -d and -D parameters. They work well in most scenarios. Using the default value of minOverlap should work fine. That will get as many reads counted as possible with sufficient confidence. For more info about the maxMOp parameter, see the users guide.

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Thank you. From my understanding, the -d and -D parameters wouldn't be implemented unless you included -P. Is that correct?

maxMOp - specify the maximum number of ‘M’ operations (matches or mis-matches) allowed in a CIGAR string. 10 by default.

I am not clear on the motivation behind this parameter. Is this to minimise the number of included insertions and deletions in the alignment?

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Yes, to use -d and -D you will have to specify -P at the same time. The maxMOp parameter limits the number of gaps in a read that are considered during read assignment.

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From your lack of comment, I assume you would also recommend to retain the default parameters for fracOverlap, fracOverlapFeature and nonOverlap?