FeatureCount for miRNA count

Question

[k.kathirvel93]

Can Anyone help me ? I want to count number of miRNA from my miRNAseq data by using featureCount. I have mapped my draft genome against HG38 by using Bowtie2. Now i want to count, so i have tried featureCounts with various parameters but i couldn't. So can anyone suggest a idea. Thanks in advance.

[WouterDeCoster]

so i have tried featureCounts with various parameters but i couldn't.

And it would be VERY helpful if you can share which parameters you tried, and what the error message was. We can't magically know what's going wrong.

[k.kathirvel93]

Command : featureCounts -t miRNA -g name -a hsa.gff -o counts.txt mapping_results.bam

Result : corrupted file ....Like ... Dot dot dot dot dot..........

[WouterDeCoster]

Please use ADD COMMENT or ADD REPLY to answer to previous reactions, as such this thread remains logically structured and easy to follow. I have now moved your post but as you can see it's not optimal. Adding an answer should only be used for providing a solution to the question asked.

It looks like your bamfile or gff is corrupted. Some things to look at:

• Are you sure that miRNA is present in your gff?
• Is 'name' present in the 9th field of your gff?
• What's the result of samtools index mapping_results.bam?

[k.kathirvel93]

Thanks for the reply WouterDeCoster

• No. gff file contains the name of the miRNA, not sequence.
• name present in 11th field
  • samtools index mapping_results.bam showing empty result. I hope the prob is with .gff file. can you suggest me from where i can get the mature microRNA .gtf file?

[WouterDeCoster]

You misunderstood my questions:

• I meant: Is the word "miRNA" present in the third field of your gff file?
• A gff file has 9 fields. Not 11 fields. Either your file is very wrong or you need to count again.
• What's the output of samtools flagstat mapping_results.bam and samtools idxstats mapping_results.bam?

[k.kathirvel93]

Thanks for the response WouterDeCoster,

1. Yes .gff file contains the name "miRNA" in third field. The .gff file was downloaded from mirbase, mature microRNA hsa.gff file.
2. /Bowtie2$ samtools flagstat TM1_filtered_Sort_PCR_RG.bam

Output :

                   109373 + 0 in total (QC-passed reads + QC-failed reads)
                   0 + 0 secondary
                   0 + 0 supplementary
                  0 + 0 duplicates
                  3793 + 0 mapped (3.47% : N/A)
                  0 + 0 paired in sequencing
                  0 + 0 read1
                  0 + 0 read2
                  0 + 0 properly paired (N/A : N/A)
                 0 + 0 with itself and mate mapped
                  0 + 0 singletons (N/A : N/A)
                 0 + 0 with mate mapped to a different chr
                  0 + 0 with mate mapped to a different chr (mapQ>=5)

[WouterDeCoster]

Again, please use ADD COMMENT or ADD REPLY to answer to previous reactions, as such this thread remains logically structured and easy to follow. I have now moved your post but as you can see it's not optimal. Adding an answer should only be used for providing a solution to the question asked.

That's quite a low fraction of mapped reads... What's in the log file of featureCounts?