Hi everyone!
I'm new to Bioinformatics. I need to write a pipeline for miRNA-Seq. The situation is the following: I have three normal samples and three cancer samples. I downloaded the stem-loop and the mature human sequences from miRBase, then I performed the mapping using Bowtie. Now I need to obtain the read counts for each sample; I'm trying to use featureCounts with mirBase hsa.gff3 file and my bam file but featureCounts doesn't work! It returns this:
Assigned 0
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_Chimera 0
Unassigned_FragmentLength 0
Unassigned_Duplicate 0
Unassigned_MultiMapping 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_NoFeatures 1831739
Unassigned_Overlapping_Length 0
Unassigned_Ambiguity 0
This is my command:
> featureCounts -t miRNA -g 'Name' -a my_path/hsa.gff3 -o my_path/my_bam_file
I have also tried with -g ID but the result is the same.
I have done a lot of researches but I don't understand where is the problem.
Thank you.
Hi, thank you for your answer!
I tried to run your command line but I had this error message:
Then I tried to run this:
and I have obtained a result like this:
hsa-miR-576-3p 22 5 0
hsa-miR-140-5p 22 15 0
hsa-miR-522-5p 22 1 0
hsa-miR-1298-5p 22 0 0
hsa-miR-133a-3p 22 1 0
hsa-miR-4743-3p 21 0 0
hsa-miR-557 23 0 0
hsa-miR-548ao-3p 22 0 0
hsa-miR-5088-5p 24 1 0
hsa-miR-4649-5p 24 0 0
hsa-miR-665 20 0 0
.....
Is it what I'm searching for?
Yes. The first column is the length. The second column is the number of reads mapped to that miRNA, the thrid column is the number of unmapped reads on that miRNA (this is a pretty meaningless number, and will normally be 0).
Hi i.sudbery,
I trust you are well during these difficult times. I was wondering if you could advise on the idxstats tool you mentioned in a previous post. I aligned my small-RNA seq to miRBase mature miRNA assembly, and naturally I now want to do further analysis. This requires read counts, but as you pointed out previously, when not aligning to the genome conventional count tools that rely on annotations struggle a bit. My question, if I have made sure my aligned BAM file only contains the reads I want (in my case unique and 1 instance of each multi-mapper), can I simply sort the BAM and use idxstats tool and use the 3rd column as the read counts for each miRNA?
Thank you for you help. Kind regards
Yes. If the BAM file is correctly filtered, then you should just be able to use the 3rd column from the output of
samtools idxstats mybam.bam
Brilliant. Thank you kindly!