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  • Incosistent number of paired end reads (BAM file generated by STAR using RSEM)

    I have used RSEM to quantify paired-end RNA-Seq data. The alignment was done
    using STAR which was called internally by RSEM, using a reference prepared using the rsem-prepare-reference script, with a GTF that I generated by downloading the GRCh38.90 gtf from Ensembl and then excluding from the GTF transcripts that are not relevant for my downstream analysis.

    I then run samtools flagstat on the genome BAM file:

    HTML Code:
    samtools flagstat SRR627536.genome.sorted.bam
    41323930 + 0 in total (QC-passed reads + QC-failed reads)
    18115150 + 0 secondary
    0 + 0 supplementary
    0 + 0 duplicates
    39343574 + 0 mapped (95.21% : N/A)
    23208780 + 0 paired in sequencing
    11604390 + 0 read1
    11604390 + 0 read2
    21228424 + 0 properly paired (91.47% : N/A)
    21228424 + 0 with itself and mate mapped
    0 + 0 singletons (0.00% : N/A)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)

    The number of mapped reads is greater then the number of paired reads. So
    if I understand correctly, I should expect that have reads for which neither the 0x1 nor the 0x4 bit are set.
    However when I run:
    HTML Code:
    samtools view -F 1 SRR627536.genome.sorted.bam
    I get a BAM file with 0 rows.

    How can this discrepancy be explained? Does STAR/RSEM interpret SAM flags in a way that is different from samtools in some way?

    Thanks in advance

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