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  • FeatureCounts and E.coli Reference

    Hi all,

    I am struggling to count Bowtie2-mapped reads using featureCounts and after three days of investigation I hope you can help me.

    My workflow is simple.
    Bacterial (E.coli) Illumina RNAseq data -> FASTQC -> Bowtie2 to custom E.coli strain reference -> Samtools subsampling -> FeatureCounts.

    Bowtie2 Mapped Reads: 34,445,043
    FeatureCounts: 2,672,611 (7.8%) Successfully assigned alignments

    The call:
    featureCounts -a reference.gtf -p -T 10 -M -O -t CDS -g ID -o output.txt mapped.bam

    Why does featureCounts only annotate 7% of the reads, even though the entire genome is full of perfectly annotated genes (see attachment) ?
    How to run featureCounts properly on bacterial RNAseq data?
    Is there an alternative software to featureCounts?

    Please have a look at the IGV attachment. Everything looks fine, I am very desperate how to debug this situation. I tried almost all combinations of featureCounts paramters. I used PROKKA and BAKTA genome annotations.

    If you have any guess or idea how to come closer to the origin of the problem please let me know.

    Here is the featureCounts output:
    ​Assigned 2672611
    Unassigned_Unmapped 0
    Unassigned_Read_Type 0
    Unassigned_Singleton 0
    Unassigned_MappingQuality 0
    Unassigned_Chimera 0
    Unassigned_FragmentLength 0
    Unassigned_Duplicate 0
    Unassigned_MultiMapping 0
    Unassigned_Secondary 0
    Unassigned_NonSplit 0
    Unassigned_NoFeatures 31772432
    Unassigned_Overlapping_Length 0
    Unassigned_Ambiguity 0​

    Best,
    Michael

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