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FeatureCount for miRNA count

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.

genome next-gen sequencing sequence software error
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written by k.kathirvel93 k.kathirvel93

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.

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written by WouterDeCoster WouterDeCoster

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

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

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written by k.kathirvel93 k.kathirvel93

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?
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written by WouterDeCoster WouterDeCoster

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?
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written by k.kathirvel93 k.kathirvel93

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?
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written by WouterDeCoster WouterDeCoster

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)
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written by k.kathirvel93 k.kathirvel93

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?

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written by WouterDeCoster WouterDeCoster

Log File contains

Status  TM1_filtered_Sort_PCR_RG.bam
Assigned    494
Unassigned_Ambiguity    0
Unassigned_MultiMapping 0
Unassigned_NoFeatures   3299
Unassigned_Unmapped 105580
Unassigned_MappingQuality   0
Unassigned_FragmentLength   0
Unassigned_Chimera  0
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_Duplicate    0
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updated by WouterDeCoster WouterDeCoster • written by k.kathirvel93 k.kathirvel93

I added markup to your post for increased readability. You can do this by selecting the text and clicking the 101010 button. When you compose or edit a post that button is in your toolbar, see image below:

101010 Button

I don't see anything wrong with the log. Just like the flagstat output: an enormous fraction of your reads are not mapped. To me, it seems that's an issue you should address first.

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written by WouterDeCoster WouterDeCoster

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