BWA-MEM: output mapped reads larger than input reads

[Kennels]

I have some bam files generated from BWA-MEM using these command:

bwa mem -t 8 ref1 R1.fa.gz R2.fa.gz | samtools view -S -h -F 4 -b - | samtools sort - bwaaln-sorted

So i should get only mapped reads in my output. I'm using bwa v0.7.5a.

Using samtools flagstat, i retrieved the number of mapped reads from the sorted bam file, and in some cases, this was higher than the number of original input paired-end reads.

Now this could be due to reads aligning to multiple places, but I don't have any "XA" tags, nor "XT" tags for that matter.

I already saw this post about getting uniquely mapping reads from BWA.
http://seqanswers.com/forums/showthread.php?t=30120

I want to get stats on unique and multi-mapping reads.
Is using MapQ=0 a reliable way of flagging multi-mapping reads?
And why am I not getting XA and XT tags at all?

Here are a couple of lines of my output - I am not seeing any XT/XA tags, nor X0 or X1 tags.

HWI-ST226:220:D0AU7ACXX:5:1306:2199:194196	83	ref1	1	58	10S78M	=	25	-54	CAGACGTGTGCTCTTCCGATCTCGTAAATTTAGTACAACCAAGGGGTAAAAATCCACACTACAACAAGCCGCTCACTATTAGACCCAA	*	NM:i:0	AS:i:78	XS:i:67
HWI-ST226:220:D0AU7ACXX:5:2206:3059:40310	163	ref1	21	0	88M	=	42	109	TAGTACAACCAAGGGGTAAAAATCCAGACTACAACAAGCCGCTCACTATTAGACCCAAATGTCCAAAAACCCTTCACAAAATCGTCAA	*	NM:i:1	AS:i:83	XS:i:83
HWI-ST226:220:D0AU7ACXX:5:1306:2199:194196	163	ref1	25	40	66M22S	=	1	54	ACAACCAAGGGGTAAAAATCCACACTACAACAAGCCGCTCACTATTAGACCCAAATGTCCAAAAACAGATCGGAAGAGCGTCGTGTAG	*	NM:i:0	AS:i:66	XS:i:66
HWI-ST226:220:D0AU7ACXX:5:2206:3059:40310	83	ref1	42	0	88M	=	21	-109	ATCCACACTACAACAAGCCGCTCACTATTAGACCCAAATGTCCAAAAACCCTTCACAAAATCGTCAATCTTCTCACTTTCCTGGCGTC	*	NM:i:0	AS:i:88	XS:i:88

thanks

[dpryan]

Regarding the read number mismatch, are you counting both mates of a pair in one case and not the other? That and multimapping are the most likely causes (I haven't a clue on the XA/XT tag issue).

[lh3]

Caused by chimeric alignments.

[Kennels]

Thanks.

But why am I not getting any XT,XA,X0 and X1 tags?
I do have some SA tags, maybe 1% of reads.
Any recommendations to extract unique and multi mapping reads? Should I just use mapQ=0?

[lh3]

Just check mapQ.

For long reads and local alignment, X0/X1 do not make much sense any more. If you want multiple mappings, use "-a".

[Kennels]

Just check mapQ.

For long reads and local alignment, X0/X1 do not make much sense any more. If you want multiple mappings, use "-a".

I tried getting all alignments with mapQ>0 and I got 214,694,901 reads (alignments), which should be unique mappings (?) since bwa only outputs the primary hit by default for mapQ > 0 (default options used here).

Just to check, i coupled the read name with the flag and counted how many uniq ones there were;
samtools view -q1 sorted.bam | awk ' { print $1"-"$2 } ' | sort | uniq | wc -l
214694478

and so there are around 420 reads which appear duplicated even with mapQ>0.
Is it possible for a read with mapQ>0 to get reported twice for some reason?

Thanks.

[Kennels]

I tried getting all alignments with mapQ>0 and I got 214,694,901 reads (alignments), which should be unique mappings (?) since bwa only outputs the primary hit by default for mapQ > 0 (default options used here).

Just to check, i coupled the read name with the flag and counted how many uniq ones there were;
samtools view -q1 sorted.bam | awk ' { print $1"-"$2 } ' | sort | uniq | wc -l
214694478

and so there are around 420 reads which appear duplicated even with mapQ>0.
Is it possible for a read with mapQ>0 to get reported twice for some reason?

Thanks.

I had a closer look at the output, and it would appear that these duplicated reads are due to chimeric reads as you mentioned Heng. I understand now what you meant. For example, the reads below were output by '-q1' option from samtools view.

HWI-ST226:220:D0AU7ACXX:5:1101:11456:146339	2193	Oa_Locus_2615_Transcript_18	2194	44	42M46H	=	2182	-54	AATTGAGCTACCAAAAACCCTAACCCAAAAATTTGTAGCGTC	*	NM:i:2	AS:i:35	XS:i:0	SA:Z:Oa_Locus_2615_Transcript_14,1923,+,21S43M24S,60,0;Oa_Locus_2615_Transcript_14,1976,-,50S38M,55,0;
HWI-ST226:220:D0AU7ACXX:5:1101:11456:146339	2193	Oa_Locus_2615_Transcript_14	1976	55	50H38M	Oa_Locus_2615_Transcript_18	2182	0	GGGGGTTTTAGTAGCTCTCTCTCTGTGGCCAGAAATGG	*	NM:i:0	AS:i:38	XS:i:0	SA:Z:Oa_Locus_2615_Transcript_14,1923,+,21S43M24S,60,0;Oa_Locus_2615_Transcript_18,2194,-,42M46S,44,2;

This the second read in a pair, but gets reported twice even though their mapQ is not 0.
I suppose using the '-M' option will flag both of these reads as secondary.

So to summarize, to get unique mappings:

1. use '-q1' option to get mapQ>0 alignments. This will include reads which are chimeric, and so will include a small level of 'multi-mapping' reads
2. from step 1, filter out reads which have "SA:" tag. This should give unique mappings in the context of bwa-mem

Is this correct?

[lh3]

That is about right. Just beware that sometimes chimeric reads are informative to break points/SVs.

[pengchy]

Hi,

still question here:
for this pair reads, i have sort the bam by the query name, they were uniquely and properply mapped. However, with 0 mapQ. According to the above mentioned method, they will be filtered for unique reads calculation.

bwa-0.7.5a mem was used here.

HISEQ700708:142:C2624ACXX:1:1101:10002:122014   99      scaffold2651    547291  0       100M    =       547537  346     ATCAAGGCCGTCATTACGGCCAGACGTGATTGTTCTGGGTACTGATTTCTCTGGATCTATACACCTAAACTGCGTGAAAATGTAGTGTAGCAATTTTAAT  CCCFFFFFHHHHHJJIHIJJJJJJJJHIIJJJIIJIIJJ?DHIGGGIJJJJJJIIJJJIJIHGHHHFFFFFFEDD@DDDDDDDFEDDDEDDDDDDDDEDD  NM:i:0  AS:i:100        XS:i:100
HISEQ700708:142:C2624ACXX:1:1101:10002:122014   147     scaffold2651    547537  0       100M    =       547291  -346    ATACAGCTAGTTTCTCCTCAGTTCGAATCGGCTTTGACGCGTTGCATGCCTCACTCTCAGTTTGATTTTTCATGAGACCATGCACTTCTTGGAATTGATC  D@:DCDDDCDDBDDCDDDDDDDEDDDDDDFFFFHHGIIJJIJJJJJIIFGJHFIIJIIJJJJJJJJJJJJJIIJIHHJJJIIIHJJJGGHHHFFFFFCCC  NM:i:0  AS:i:100        XS:i:100

[pengchy]

I understand this, there only output one record for multiple mapped reads.

Hi,

still question here:
for this pair reads, i have sort the bam by the query name, they were uniquely and properply mapped. However, with 0 mapQ. According to the above mentioned method, they will be filtered for unique reads calculation.

bwa-0.7.5a mem was used here.

HISEQ700708:142:C2624ACXX:1:1101:10002:122014   99      scaffold2651    547291  0       100M    =       547537  346     ATCAAGGCCGTCATTACGGCCAGACGTGATTGTTCTGGGTACTGATTTCTCTGGATCTATACACCTAAACTGCGTGAAAATGTAGTGTAGCAATTTTAAT  CCCFFFFFHHHHHJJIHIJJJJJJJJHIIJJJIIJIIJJ?DHIGGGIJJJJJJIIJJJIJIHGHHHFFFFFFEDD@DDDDDDDFEDDDEDDDDDDDDEDD  NM:i:0  AS:i:100        XS:i:100
HISEQ700708:142:C2624ACXX:1:1101:10002:122014   147     scaffold2651    547537  0       100M    =       547291  -346    ATACAGCTAGTTTCTCCTCAGTTCGAATCGGCTTTGACGCGTTGCATGCCTCACTCTCAGTTTGATTTTTCATGAGACCATGCACTTCTTGGAATTGATC  D@:DCDDDCDDBDDCDDDDDDDEDDDDDDFFFFHHGIIJJIJJJJJIIFGJHFIIJIIJJJJJJJJJJJJJIIJIHHJJJIIIHJJJGGHHHFFFFFCCC  NM:i:0  AS:i:100        XS:i:100