After alignment using BWA, I have removed the dupliment using the samtools(Version: 1.9).
My procedure is as follows:
bwa mem -k 32 -M ref.fa read1 read2 > out.sam samtools view -@ 0 -b -T ref.fa -o out.bam in.sam samtools sort -n -o out.nameSrt.bam in.bam samtools fixmate -r -m in.nameSrt.bam out.fixmate.bam samtools sort -o out.fixmate.sort.bam in.fixmate.bam samtools markdup -r -S -s in.fixmate.sort.bam out.markdup.bam samtools flagstat in.markdup.bam > out.markdup.flagstat
The flagstat output result is as follows:
21611397 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 21611397 + 0 mapped (100.00% : N/A) 21422330 + 0 paired in sequencing 10711165 + 0 read1 10711165 + 0 read2 19797684 + 0 properly paired (92.42% : N/A) 21422330 + 0 with itself and mate mapped 0 + 0 singletons (0.00% : N/A) 1306000 + 0 with mate mapped to a different chr 727043 + 0 with mate mapped to a different chr (mapQ>=5)
Why is the total read number still more than the paired in sequencing after removing the duplicate in samtools flagstat output? Is there anything wrong with my procedure?