BWA mapping quality 0 for all reads

Question

[OhHiImNewHere]

I've mapped my reads using BWA but when I looked at them on IGV, they all have a mapping quality of 0. All of them. I don't think they have multiple possible matches because when I have Blasted a handful, they all have only one match. I think they are mapped correctly because when I look at my polymorphisms, they all match known polymorphisms in the database.

But obviously I cannot do any variant calling on reads with a mapping quality of 0.

This is the script I have used:

bwa mem -t 16 $path/to/ref_genome.fa $path/to/s1_S1100619_1P.fq.gz $path/to/s1_S1100619_2P.fq.gz > s1_S1100619_aln_pe.sam

Any ideas why this might have happened?

[Istvan Albert]

Show a few lines of your SAM file, there is other information in the SAM file that can help tracking down the reasons that an alignment is reported a certain way.

[OhHiImNewHere]

   A00627:159:HWYY5DSXY:4:1101:27715:7874   81  chrX    108691208   0   150M    =   108691132   -226    AGACTAGCCACACGTCAAGTGCTGAGTAATTATTTGTGGCTTGTGGCTACCATGTTGGACAGGGCAGCAAAAGAAGAGGATACTCCTTACCTTACTGCCAAAGTGGGGATTGATAAACGTTACATCCTCCAAAGTCAGTAGAATTCTATT  FFF,FF,F:FFFFFF,FFFFFFFFF:F,FFFFF:FFFFFF:FFFFF:F::FFFFFFFF:FFFF,FFFFFFF:FFFFFFFF:FFFF:FFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF:F  NM:i:0  MD:Z:150    AS:i:150    XS:i:150    XA:Z:chrX,-108691208,150M,0;
    A00627:159:HWYY5DSXY:4:1101:27715:7874  161 chrX    108691132   0   150M    =   108691208   226 AATAAGATATTTTACATGTTTTTATACTGTTTTCTAAACCCACTGAGTACTTTACTCTTATAGCACATCACAATTCAGACTAGCCACACGTCAAGTGCTGAGTAATTATTTGTGGCTTGTGGCTACCATGTTGGACAGGGCAGCAAAAGA  FFFFFFFFFF:FFFFF:FFF,FFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FF:FFFFFFFFFFFF:FF:FFFFFFFFFFFFFFFFFFF:FFF:F:FFFFFF:FF:FFFFFFFF  NM:i:0  MD:Z:150    AS:i:150    XS:i:150    XA

[Istvan Albert]

See the XA tags there in the first alignment? That tag indicates that the read has a secondary alignment on chrX at position 108691208 (reverse):

XA:Z:chrX,-108691208,150M,0;

check whether this is a universal problem.

[Istvan Albert]

Actually, I just realized this is the same as the position reported for the main alignment - that kind of makes no sense. Is it possible that you have chrX listed twice in your reference genome? Or the indexing was done incorrectly?

[OhHiImNewHere]

Yes, it looks as though they are all listed for the same position twice. I wonder if it because I have accidentally put two reference genomes in the same directory???

[OhHiImNewHere]

A00627:159:HWYY5DSXY:4:1101:2465:1031   97      chr11   3681333 0       150M    =       3681361 178     TNAGGCTGGGGGGCTTTGCTTCTGCCTCCCTGAAGCATGTTGCAGCCCAGCAGTTTGGTGAGGACACCTTCTTCGGCATCTGGACCTGCCTTGGGGCCCCTATCAAGGGCTACTCCTTCTTCCCTGGAGAGGAAGAGGTGCTGATCCCCC  F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFF,FFFFFFFFF:FFFFFF  NM:i:1  MD:Z:1G148      AS:i:148        XS:i:148        XA:Z:chr11,+3681333,150M,1;
A00627:159:HWYY5DSXY:4:1101:2465:1031   145     chr11   3681361 0       150M    =       3681333 -178    CCTGAAGCATGTTGCAGCCCAGCAGTTTGGTGAGGACACCTTCTTCGGCATCTGGACCTGCCTTGGGGCCCCTATCAAGGGCTACTCCTTCTTCCCTGGAGAGGAAGAGGTGCTGATCCCCCCCTTTGAGACCTTCCAAGTGATCAATGC  FFFF,FFFFFFFF,FFFFFFFFFF:FFFFF,FF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF  NM:i:0  MD:Z:150        AS:i:150        XS:i:150        XA:Z:chr11,-3681361,150M,0;
A00627:159:HWYY5DSXY:4:1101:5050:1031   97      chr14   95080797        0       150M    =       95080852        205     CNCCTGGCTCTGGGGCTCTTGGCGGCTGGGTTCTGCCCTGCTGTCCTCTGCCACCCTAACAGCCCACTTGACGAGGAGAATCTGACCCAGGAGAACCAAGACCGAGGGACACACGTGGACCTCGGATTAGCCTCCGCCAACGTGGACTTC  F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F  NM:i:1  MD:Z:1T148      AS:i:148        XS:i:148        XA:Z:chr14,+95080797,150M,1;
A00627:159:HWYY5DSXY:4:1101:5050:1031   145     chr14   95080852        0       150M    =       95080797        -205    CTAACAGCCCACTTGACGAGGAGAATCTGACCCAGGAGAACCAAGACCGAGGGACACACGTGGACCTCGGATTAGCCTCCGCCAACGTGGACTTCGCTTTCAGCCTGTACAAGCAGTTAGTCCTGAAGGCCCCTGATAAGAATGTCATCT  FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFF  NM:i:0  MD:Z:150        AS:i:150        XS:i:150        XA:Z:chr14,-95080852,150M,0;

[Istvan Albert]

Every alignment is reported as a multiple alignment to itself. Indicates some sort of data problem

Answer 1

[Istvan Albert]

Rerun your indexing but on a file that contains a single copy of each chromosome.

From the above data, it looks like your reference genome has been accidentally duplicated.