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Old 12-21-2012, 12:58 AM   #1
manore
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Location: paris

Join Date: Jun 2011
Posts: 19
Default Bowtie2 insert size=0

Hi,
I used dwgsim to create simulated read pairs.
Then, I aligned these read pairs to the reference genome with Bowtie2.
I obtained 5063956 reads pairs with insert size =0
Code:
samtools view file.bam | awk '{ if ( $9 ==0){print $0;} }' | wc -l
5063956

What does it means?

IGV uses color coding to flag anomalous insert sizes. When I look these read pairs with insert size=0, it appear in grey. In IGV grey is for inserts that are like than expected.


This the flagstat result (file.bam):
10022022 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
9979404 + 0 mapped (99.57%:-nan%)
10022022 + 0 paired in sequencing
5011011 + 0 read1
5011011 + 0 read2
4958066 + 0 properly paired (49.47%:-nan%)
8789364 + 0 with itself and mate mapped
1190040 + 0 singletons (11.87%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

This is the results I obtain according to the insert size column (file.bam):
Mapped in correct orientation and within insert size (i.e properly paired) : 4958066
Mapped uniquely but wrong insert size : 3831298
Both unmapped : 38188
One of the mate is unmapped : 1194470
Mapped within the insert size but wrong orientation: 0


I notice that 3831298+38188+1194470=5063956, it correspond reads pairs with insert size =zero

Someone can help me?
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Old 12-21-2012, 08:45 AM   #2
westerman
Rick Westerman
 
Location: Purdue University, Indiana, USA

Join Date: Jun 2008
Posts: 1,104
Default

So ... what is your question? You answered why you came up with 5063956 zero insert size reads -- they correspond to reads that (a) Mapped uniquely but wrong insert size, (b)Both unmapped or (c) One of the mate is unmapped. Are you expecting a different value than 0 in that insert size field? The sam/bam manual says:

Quote:
TLEN: signed observed Template LENgth. If all segments are mapped to the same reference, the
unsigned observed template length equals the number of bases from the leftmost mapped base
to the rightmost mapped base. The leftmost segment has a plus sign and the rightmost has a
minus sign. The sign of segments in the middle is undefined. It is set as 0 for single-segment
template or when the information is unavailable.
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Old 12-26-2012, 01:03 AM   #3
manore
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Location: paris

Join Date: Jun 2011
Posts: 19
Default

Thanks for your response. My question was, is it possible to have read pairs with insert size=0? and how it is possible.
The insert size expected is about 560 pb.

*****
According to the sam/bam manual "It is set as 0 for single-segment template or when the information is unavailable." So it would like to say that for a couple of reads, insert size can be zero because one read mapp and the other not (i.e One of the mate is unmapped) or because the information is unavailable.

According to my analyse, zero insert size reads corresponds to reads that (a) Mapped uniquely but wrong insert size, (b)Both unmapped AND (c) One of the mate is unmapped.

It's actually not clear to me how to interpret such a situation and why Bowtie2 produces this kind of paired alignments
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