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  • Question: counting after aligning miRNA reads to miRBase mature miRNA instead of refe

    I have human miRNA-seq data. In the past I have aligned these data using Bowtie2 to the reference genome (Homo_sapiens.GRCh38.dna.toplevel.fa), and I have subsequently performed counting using featureCounts with the annotation file hsa.gff3 from miRBase. . Now I have aligned the reads to the mature miRNA from miRBase (mature.fa), but when I look at the resulting bam files, the reads have a flag of 4 (segment unmapped) and there is no position information in mature.fa.

    So can I use mature.fa as the reference for alignment and if so how is counting performed?

    Thanks, Ina

  • #2
    Hi Ina,

    I use samtools and picard-tools to get the counts for miRNAs.

    Here is what I would do for bowtie2 alignment to miRBase
    bowtie2 -L8 --local -x miRBase-mature-hsa-index -U FILE.fastq -S FILE.sam
    samtools view -b -S FILE.sam > FILE.bam
    samtools sort FILE.bam > FILE.sorted
    picard-tools BuildBamIndex I=FILE.sorted
    picard-tools BamIndexStats I=FILE.sorted > FILE.txt

    Makes sense?

    Cheers,
    sergio

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