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

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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

[sbarberan]

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