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  • Can or should we perform feature counting with `bedtools intersect -c`?

    I have the following BAM file and a annotation file.
    What I want to do is to calculate reads count in
    I can do it with the following three methods.


    Below is the code

    Code:
        #!/bin/bash
        INPUT_BAM=my_paired_end_alignment.bam
        ANNNOT_BED=peak_annot.bed
        ANNNOT_GTF=peak_annot.gtf
        
        # Temporary file
        ALIGNMENT_BED=my_paired_end_alignment.bed
        
        #-----------
        # Method 1
        #-----------
        FINAL_COUNT_METHOD1=output_method1.txt
        bedtools bamtobed -bedpe -i $INPUT_BAM > $ALIGNMENT_BED
        bedtools intersect -a $ANNOT_BED -b $ALIGNMENT_BED -c > $FINAL_COUNT_METHOD1
        
        #-----------
        # Method 2
        #-----------
        FINAL_COUNT_METHOD2=output_method2.txt
        bedtools multicov -bams $INPUT_BAM -bed $ANNOT_BED > $FINAL_COUNT_METHOD2
        
        #-----------
        # Method 3
        #-----------
        featureCounts  -a $ANNOT_GTF -o counts.txt $INPUT_BAM
    The result given by Method1 is so different from Method2 (totally uncorrelated),
    whereas Method2 is highly correlated with Method3.

    My question is Method1 the right way to count feature? If not why?
    Should we use Method2 or Method3 instead?

  • #2
    For reference cross-posted: https://www.biostars.org/p/270366/

    Comment

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