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Multi-mapping High with featureCounts but not STAR
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18 months ago
garbuzov • 0

Hi everyone, I am sequencing neurons isolated from rat. I'm working with a limited amount of material, so using Nugen Ovation Universal library kits. The QC using FastQC looks ok. My library is paired-end with 75bp reads. I ran STAR using this command:

STAR --runThreadN 3 \
--genomeDir ../genomes/rn6/Ensembl/star2 \
--readFilesCommand gunzip -c \
--readFilesIn ${R1} ${R2} \
--outFileNamePrefix starMapped/${job_name} \
--outSAMtype BAM Unsorted \
--seedSearchStartLmax 40 \
--outFilterScoreMinOverLread 0.5 \
--outFilterMatchNminOverLread 0.5

My unique mapped reads is ~70-90% for all samples with multi-mapped reads 7-15%.

I used the same GTF file to then make the count table using featureCounts using this command:

featureCounts -T 6 -p -t exon -g gene_name -a ../genomes/rn6/Ensembl/Rattus_norvegicus.Rnor_6.0.93.gtf -o combined_counts.txt *.bam

But my % assigned reads is only 33% and unassigned_multimap reads are ~30% of my reads and unassigned_NoFeature are the other third. I don't understand how the number of multi-mapping reads and mapping can be so different between the two packages given I am using the same GFT file. What's going on here? And why is the number of unassigned reads so high with featureCounts?

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Can you confirm that you are using the BAM files generated by STAR for counting with featureCounts? If you can post detailed STAR stats that would be great.

Since you did not change the -outFilterMultimapNmax parameter for STAR it is set to 10 (default) so STAR is reporting up to 10 alignments per such reads. Those will not be counted by featureCounts unless you turn the -M option on.

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You can obtain counts directly from STAR with --quantMode GeneCounts, this quantification should be similar to that obtained with featureCounts. You didn't use -s in your featureCounts command, are you sure the Nugen Ovation results in an unstranded library?

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Good point (see this, under Data analysis) :

Note that in libraries generated by the Ovation Universal RNA-Seq System, the forward read corresponds to the sense strand.

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