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  • Why Is My Uniquely Mapped Read Count in Samtools Higher Than in Bowtie2?

    Hi everyone,

    I aligned my paired-end sequencing data using Bowtie2 and processed the resulting BAM files with Picard MarkDuplicates. However, I noticed a discrepancy between Bowtie2’s uniquely mapped read count and Samtools’ uniquely mapped reads after filtering. Bowtie2 alignment statistics (bowtie2.txt):


    8536975 reads; of these: 5180282 (60.68%) aligned concordantly 0 times (unmapped) 1868933 (21.89%) aligned concordantly exactly 1 time (uniquely mapped) 1487760 (17.43%) aligned concordantly >1 times (multi-mapped) Overall alignment rate: 39.32%
    • Uniquely mapped reads reported by Bowtie2: 1,868,933
    Samtools read count after removing duplicates (samtools view -c -q 30 CR05NFYA_S1_mdu.bam):

    2,877,386
    • Samtools reports ~1M more uniquely mapped reads than Bowtie2.
    Questions:
    1. Why does my final _mdu.bam file (uniquely mapped, deduplicated) contain ~1M more reads than Bowtie2’s unique count?
    2. Does Bowtie2 apply stricter filtering than samtools view -q 30 when classifying uniquely mapped reads?
    3. Could multi-mapped reads or soft-clipped alignments still be counted in _mdu.bam, even with MAPQ ≥ 30?
    4. How does paired-end read counting differ between Bowtie2 and Samtools? Does Bowtie2 exclude some properly paired reads?
    What I Have Tried:
    • Verified read counts before and after duplicate marking:bash
      CopyEdit
      samtools view -c -F 1024 CR05NFYA_S1_mdu.bam
    • Checked MAPQ distribution:bash
      CopyEdit
      samtools view CR05NFYA_S1_mdu.bam | awk '{print $5}' | sort -n | uniq -c
    • Excluded secondary alignments:bash
      CopyEdit
      samtools view -c -F 256 CR05NFYA_S1_mdu.bam

    I would appreciate any insights!

    Thanks in advance!

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