The discordant and mixed reads identification in Bowtie2

[daisyko]

Hi all,

I have tried to align my FASTQs (PE; 150bp) with a spike-in sequence of 300 bp and I padded 100 bp of "N" in the beginning and in the end of the spike-in sequence to build the reference index.

Aligning with or without "--no-mixed" and "--no-discordant" parameters, the alignment results were different. Below I showed an example of Bowtie2 recognizing a read pair as discordant and mixed. However, from the RNAME fields of BAM records, the "discordant" or "mixed" alignment results are actually aligning to the same reference genome (the DNA_spike_in). And also, they looked quite "properly paired" to me as they were actually mapping to the two ends of the reference. May I know why this kind of reads were unable to obtain a "properly paired" result? Moreover, may I know if there is a way to get rid of this kind of issue by still enabling the--no-mixed, --no-discordantoption? Thanks a lot!

Bowtie2 (--no-mixed, --no-discordant)

A00549:407:HM3LVDSXC:3:1101:18304:1031  77      *       0       0       *       *       0       0       ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGACT       FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF     YT:Z:UP RG:Z:SNAME
A00549:407:HM3LVDSXC:3:1101:18304:1031  141     *       0       0       *       *       0       0       TCGTCGATGGGTTCGGGAATGCAGGATATTCCCACCTCCGGTTAAGGATGGAAGAGAGTATCTGTTCCACGAATCAGCGGTAAAGGTTGACTTAAATCGACCAGTAACAGGTGGCCTTTTGAAGAGGATCAGAAATGGGAAGAAGGCGAAG       FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFF:F:FFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFF     YT:Z:UP RG:Z:SNAME

Bowtie2 ( --no-discordant)

A00549:407:HM3LVDSXC:3:1101:18304:1031  97      DNA_spike_in    81      42      151M    =       230     0       ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGACT    FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF        AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:151        YT:Z:UP RG:Z:SNAME
A00549:407:HM3LVDSXC:3:1101:18304:1031  145     DNA_spike_in    230     42      151M    =       81      0       CTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAAGGCCACCTGTTACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGGAACAGATACTCTCTTCCATCCTTAACCGGAGGTGGGAATATCCTGCATTCCCGAACCCATCGACGA    FFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFF:F:FFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF        AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:151        YT:Z:UP RG:Z:SNAME

Bowtie2 (--no-mixed)

A00549:407:HM3LVDSXC:3:1101:18304:1031  97      DNA_spike_in    81      42      151M    =       230     300     ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGACT    FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF        AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:151        YS:i:0  YT:Z:DP RG:Z:SNAME
A00549:407:HM3LVDSXC:3:1101:18304:1031  145     DNA_spike_in    230     42      151M    =       81      -300    CTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAAGGCCACCTGTTACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGGAACAGATACTCTCTTCCATCCTTAACCGGAGGTGGGAATATCCTGCATTCCCGAACCCATCGACGA    FFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFF:F:FFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF        AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:151        YS:i:0  YT:Z:DP RG:Z:SNAME

[ch4rr0]

Hello,

I was unable to replicate this one. I tried with the following inputs but in the end got a concordant alignment, as expected. Would it be possible to share some sample input that I can use to recreate this issue?

Reference:

>spike
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGA
CTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAAGGCCACCTGTTACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGGAACAGATACTCTCTTCCATCCTTAACCGGAGGTGGGAATATCCTGCATTCCCGAACCCATCGACGA
NNNNNNNNNNNNNNNNNNNN

Read 1:

@r1
ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGACT
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

Read 2:

@r1
TCGTCGATGGGTTCGGGAATGCAGGATATTCCCACCTCCGGTTAAGGATGGAAGAGAGTATCTGTTCCACGAATCAGCGGTAAAGGTTGACTTAAATCGACCAGTAACAGGTGGCCTTTTGAAGAGGATCAGAAATGGGAAGAAGGCGAAG
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

./bowtie2-align-s -x spike -1 spike_1.fq -2 spike_2.fq
1 reads; of these:
  1 (100.00%) were paired; of these:
    0 (0.00%) aligned concordantly 0 times
    1 (100.00%) aligned concordantly exactly 1 time
    0 (0.00%) aligned concordantly >1 times
    ----
    0 pairs aligned concordantly 0 times; of these:
      0 (0.00%) aligned discordantly 1 time
    ----
    0 pairs aligned 0 times concordantly or discordantly; of these:
      0 mates make up the pairs; of these:
        0 (0.00%) aligned 0 times
        0 (0.00%) aligned exactly 1 time
        0 (0.00%) aligned >1 times
100.00% overall alignment rate
@HD	VN:1.0	SO:unsorted
@SQ	SN:spike	LN:400
@PG	ID:bowtie2	PN:bowtie2	VN:2.4.1	CL:"/bowtie2/bowtie2-align-s -x spike -1 spike_1.fq -2 spike_2.fq"
r1	99	spike	81	42	151M	=	230	300	ATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAATCCCGGCGCTCATGACT	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:151	YS:i:0	YT:Z:CP
r1	147	spike	230	42	151M	=	81	-300	CTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAAGGCCACCTGTTACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGGAACAGATACTCTCTTCCATCCTTAACCGGAGGTGGGAATATCCTGCATTCCCGAACCCATCGACGA	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	AS:i:0	XN:i:0	XM:i:0	XO:i:0	XG:i:0	NM:i:0	MD:Z:151	YS:i:0	YT:Z:CP