FeatureCounts

Question

[beginner123]

Hi

My assigned reads don't get higher if I lower my mapping score. Can anyone know what I must do to get a higher amount of reads?

featureCounts \
    -Q 10 \
    -g gene_name \
    -a /mnt/storage/data/resources/genomes/hg38/hg38.refGene.agat.gtf \
    -o all.counts \
    C1.bam C2.bam S1.bam S2.bam

[ATpoint]

You have more than half of the reads being multimappers, hence fiddling with mapping quality is not going to help. What is the context here, do you have any idea from the wetlab side why that is? Is this something like short/small RNA-seq or is the read length exceptionally short?

[beginner123]

Status  C1.bam  C2.bam  S1.bam  S2.bam
Assigned    13794555    14166283    13113126    12831852
Unassigned_Unmapped 0   0   0   0
Unassigned_MappingQuality   0   0   0   0
Unassigned_Chimera  0   0   0   0
Unassigned_FragmentLength   0   0   0   0
Unassigned_Duplicate    0   0   0   0
Unassigned_MultiMapping 19433175    16759053    16007608    22902234
Unassigned_Secondary    0   0   0   0
Unassigned_Nonjunction  0   0   0   0
Unassigned_NoFeatures   1606056 1623631 1637217 1694758
Unassigned_Overlapping_Length   0   0   0   0
Unassigned_Ambiguity    575965  569557  718212  596196

[beginner123]

I'm doing bulk RNA sequencing

[ATpoint]

Cannot help without proper details, put some effort. RNA-seq is very standard these days, probably your library is poor quality.