<p>Bwa-mem may produce chimeric alignment, where different parts of a read are mapped to distinct loci. flagstat counts them as two reads.</p>

CC BY-SA 4.0
2018-11-19T14:14:54.783Z
2018-11-19T14:14:54.783Z
user172818
<p>After alignment using BWA, I have removed the dupliment using the samtools(Version: 1.9).</p>

<p>My procedure is as follows:</p>

<pre><code>bwa mem -k 32 -M ref.fa read1 read2 &gt; 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 &gt; out.markdup.flagstat
</code></pre>

<p>The flagstat output result is as follows:</p>

<pre><code>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&gt;=5)
</code></pre>

<p>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?</p>

<ngs><samtools>
Why is the total read number still more than the paired in sequencing after removing the duplicate in samtools flagstat output?
# Why is the total read number still more than the paired in sequencing after removing the duplicate in samtools flagstat output?

(Asked by: username_0 on 2018-11-19)

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?

**Tags:** ngs, samtools

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## 1 answers

### Answer by username_1 on 2018-11-19. Score: 2 | Accepted answer

Bwa-mem may produce chimeric alignment, where different parts of a read are mapped to distinct loci. flagstat counts them as two reads.


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