featurecounts multi-map and multi-overlap modes
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Camille Daniels
Oct 12, 2014, 8:40:24 AM10/12/14
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Hello,
I've run featurecounts (subread-1.4.5-p1-Linux-x86_64) using a bam file containing paired-end DNA reads mapped to an annotated bacterial genome.
Total fragments: 832927
Running ./featureCounts -T 6 -p -B -M -R -t CDS -g ID -a bact.gff -o s1_CDS_multimap.txt s1_sortcoord.bam produces
I've run featurecounts (subread-1.4.5-p1-Linux-x86_64) using a bam file containing paired-end DNA reads mapped to an annotated bacterial genome.
Total fragments: 832927
Running ./featureCounts -T 6 -p -B -M -R -t CDS -g ID -a bact.gff -o s1_CDS_multimap.txt s1_sortcoord.bam produces
Assigned 714609
Unassigned_Ambiguity 97850
Unassigned_MultiMapping 0
Unassigned_NoFeatures 20468
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_FragementLength 0
Unassigned_Chimera 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_Duplicate 0
and sum of counts in output txt file is : 714609
When I add -O option to the command above to also consider multi-overlaps, the count sum in the output file below does not match the total assigned.
Is this correct? Are the counts inflated due to reads that may be overlapping more than one meta-feature?
Assigned 812459
Unassigned_Ambiguity 0
Unassigned_MultiMapping 0
Unassigned_NoFeatures 20468
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_FragementLength 0
Unassigned_Chimera 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_Duplicate 0
Sum of counts in txt file 1,098,102
Unassigned_Ambiguity 97850
Unassigned_MultiMapping 0
Unassigned_NoFeatures 20468
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_FragementLength 0
Unassigned_Chimera 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_Duplicate 0
and sum of counts in output txt file is : 714609
When I add -O option to the command above to also consider multi-overlaps, the count sum in the output file below does not match the total assigned.
Is this correct? Are the counts inflated due to reads that may be overlapping more than one meta-feature?
Assigned 812459
Unassigned_Ambiguity 0
Unassigned_MultiMapping 0
Unassigned_NoFeatures 20468
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_FragementLength 0
Unassigned_Chimera 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_Duplicate 0
Sum of counts in txt file 1,098,102
Wei Shi
Oct 12, 2014, 9:29:21 PM10/12/14
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Dear Camille Daniels,
Yes, this is expected. When you used the '-O' option, each read will be assigned to all their overlapping features and the resulting number of counts should be equal to or greater than the number of assigned reads. In your case, when you turned on '-O', you got 812459 reads that were successfully assigned but the total number of counts (1,098,102) is greater than the number of assigned reads since quite a few reads were counted multiple times.
In your first run of featureCounts summarization where '-O' was not used, 97850 reads were found to overlap with more than one gene and these reads were not assigned due to assignment ambiguity. 714609 reads were successfully assigned and each of these reads was assigned to only one gene. These reads plus the 97850 unassigned reads constitute the 812459 reads that were assigned in your second run where '-O' was turned on.
Hope this makes it clear.
Best regards,
Wei
Yes, this is expected. When you used the '-O' option, each read will be assigned to all their overlapping features and the resulting number of counts should be equal to or greater than the number of assigned reads. In your case, when you turned on '-O', you got 812459 reads that were successfully assigned but the total number of counts (1,098,102) is greater than the number of assigned reads since quite a few reads were counted multiple times.
In your first run of featureCounts summarization where '-O' was not used, 97850 reads were found to overlap with more than one gene and these reads were not assigned due to assignment ambiguity. 714609 reads were successfully assigned and each of these reads was assigned to only one gene. These reads plus the 97850 unassigned reads constitute the 812459 reads that were assigned in your second run where '-O' was turned on.
Hope this makes it clear.
Best regards,
Wei
Camille Daniels
Oct 13, 2014, 9:41:21 AM10/13/14
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Wei,
Yes-- that helps!
Thank you!
Camlle
Yes-- that helps!
Thank you!
Camlle
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