Answers.Body: <p>If a fragment overlaps multiple genomic features then it's unassignable, which is the appropriate way to handle ambiguous mappings when the resulting counts are then given to tools like DESeq2.</p>
  
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Answers.CreationDate: 2019-03-18T10:44:11.987Z
Answers.Text: fair enough, so I will not use the -O option, thanks
Answers.UserDisplayName: Biomagician
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Answers.CreationDate: 2019-03-18T08:12:53.997Z
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Answers.UserDisplayName: Devon Ryan
Body: <p>I am using <a href="https://academic.oup.com/bioinformatics/article/30/7/923/232889" rel="nofollow noreferrer">featureCounts</a> from the subread package to count the number of fragments per gene in an RNA-seq experiment. I feed it a BAM file with paired-end reads aligned to a reference genome. In the help page of the executable, which I have installed using <code>conda install subread</code>, there is a section about overlaps, including for example the <code>-O</code> option which will assign the reads/fragments to all overlapping features. What is the default behaviour? Assigning to reads/fragments to the feature with largest overlap? not assigning multi-overlapping reads/fragments?</p>
  
  <p>I would appreciate your help on this.</p>
  
  <p>OS: macOS X</p>
  
  <p>featureCounts version: 1.6.3</p>
  
Tags: <software-usage><subread><featurecounts>
Title: How do I tell featureCounts to ignore fragments that overlap with more than one feature?
ThreadText: # How do I tell featureCounts to ignore fragments that overlap with more than one feature?
  
  (Asked by: username_0 on 2019-03-18)
  
  I am using [featureCounts](https://academic.oup.com/bioinformatics/article/30/7/923/232889) from the subread package to count the number of fragments per gene in an RNA-seq experiment. I feed it a BAM file with paired-end reads aligned to a reference genome. In the help page of the executable, which I have installed using `conda install subread`, there is a section about overlaps, including for example the `-O` option which will assign the reads/fragments to all overlapping features. What is the default behaviour? Assigning to reads/fragments to the feature with largest overlap? not assigning multi-overlapping reads/fragments?
  
  I would appreciate your help on this.
  
  OS: macOS X
  
  featureCounts version: 1.6.3
  
  **Tags:** software-usage, subread, featurecounts
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2019-03-18. Score: 1 | Accepted answer
  
  If a fragment overlaps multiple genomic features then it's unassignable, which is the appropriate way to handle ambiguous mappings when the resulting counts are then given to tools like DESeq2.
  
  > **Comments:**
  > **username_0**: fair enough, so I will not use the -O option, thanks  
  
  ---
  
  