Bowtie2 Output, Concordant Vs. Discordant Mapping??

Question

[mengqingying]

Here are several lines from the sam file obtained from running bowtie2. Could anybody tell me, how to tell if the mapping is concordant or discordant mapping? Many thanks!!

HWI-ST1148:86:C1PTAACXX:3:2316:20566:100830 81 chr2 262787705 44 100M chr1 177401969 0
GTGAGTGTAGCACAGGCGCTCACCAAAGTTCCCTTATTTGGACATCAGAGGGAATTGTCAGTCGGGTTTTTACTTAAATTAGGTAGATCTTTCTTGAATT 
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HWI-ST1148:86:C1PTAACXX:3:2316:20566:100830 161 chr1 177401969 44 76M24S chr2 262787705 0 GCCACACTGCTGCGTCCTGGGGCCAGCTCCTCCTCAGTCATCTCTACCCAGCCTAGGGAGCGCACAGCGAAACACTTGATCTATTAAGTTATTAATATGG 
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HWI-ST1148:86:C1PTAACXX:3:2316:20514:100769 83 chrX 11366080 44 76M = 11366080 -76 GTCTTGCACAATTAATACTGTCTTTGTCACCATCCAAAACACACAACGTGTCATAAGAGGCTTTCAAACGATCAAA 
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HWI-ST1148:86:C1PTAACXX:3:2316:20514:100769 163 chrX 11366080 44 76M = 11366080 -76 GTCTTGCACAATTAATACTGTCTTTGTCACCATCCAAAACACACAACGTGTCATAAGAGGCTTTCAAACGATCAAA 
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HWI-ST1148:86:C1PTAACXX:3:2316:20986:100818 83 chr10 109113908 1 4M1I95M = 109113853 -154 AGGGAAAAAAACCAGAACAGAAACTGGGTCCTAAGGCTCGGACTTTCCCACCCTGTTAGACTGGCAAGAAAAGAAGTGGTAACTAGTTCAGGAACCCTTG 
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HWI-ST1148:86:C1PTAACXX:3:2316:20986:100818 163 chr10 109113853 1 59M1I40M = 109113908 154 GGTGTCGATGCAAACGTTCCCAACTCAAGGCAAGTAACAACCCATGGAGGTCAGGAGGGAAAAAAACCAGAACAGAAACTGGGTCCTAAGGCTCGGACTT 
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HWI-ST1148:86:C1PTAACXX:3:2316:19071:100776 99 chr1 86417046 11 100M = 86417078 107 ACTGGGGCAGAGATGACACCATGAGAAAGAGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGCGCG 
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HWI-ST1148:86:C1PTAACXX:3:2316:19071:100776 147 chr1 86417078 11 75M = 86417046 -107 TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGCGCGTGCGCGC 
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Answer 1

[Philipp Bayer]

What's your expected insert-size? (You need that to know whether a pair is discordant)

Pretty sure the first pair is discordant, because both reads align on different chromosomes.

The second pair is apparently concordant because both reads are identical and align on the same position, from the help:

Bowtie 2's default behavior is to consider overlapping and containing as being consistent with concordant alignment.

See Bowtie's help for a longer explanation of discordant vs. concordant.

Answer 2

[mengqingying]

we targeted for 100PE, but as there was something wrong in the library prep, the final fragment size is around 125, thus there is about ~75 overlapping on average :(

Answer 3

[ohadg123]

You can tell it from the bitwise FLAG of the SAM format.

It's the first number after the read's name.

In your data, it's 83 for this read:

HWI-ST1148:86:C1PTAACXX:3:2316:20514:100769    83    chrX 11366080 44 76M = 11366080 -76

You can interpret the flag's number/meaning here: http://broadinstitute.github.io/picard/explain-flags.html

On this link, the concordant mapping flag is (2) "read mapped in proper pair"

Answer 4

[Ekalavya]

A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints (--fr/--rf/--ff, -I, -X).

--fr/--rf/--ff: The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand.

-I: The minimum fragment length for valid paired-end alignments.

-X: The maximum fragment length for valid paired-end alignments.

source: Galaxy-Bowtie2 options