Hi,
I received some bam files prepared elsewhere in the past and I want to
re-create the VCF files. From the header I know human_g1k_v37.fasta.gz used to
be used as the reference by bwa aligner. Also seems last time it was processed
using MarkDuplicates.
There is
ftp.ncbi.nlm.nih.gov/1000genomes/ftp/technical/reference/human_g1k_v37.fasta.gz
and also ftp://ftp.broadinstitute.org/bundle/2.8/b37/human_g1k_v37.fasta.gz
file with the same name. I picked the former origin.
I executed now:
samtools calmd -Arb dedup.realigned.bam
ftp.ncbi.nlm.nih.gov/1000genomes/ftp/technical/reference/human_g1k_v37.fasta.gz
> dedup.realigned.calmd.bam
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:1106:17285:41358': 15 -> 70
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:2312:4142:89740': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:2216:4498:85887': 0 -> 2
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:2312:4142:89740': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:1213:15077:9565': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:2216:4498:85887': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:1213:12195:85510': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:1213:15077:9565': 0 -> 1
[bam_fillmd1] different NM for read
'HWI-xxxxx:xxx:xxxxxxxxx:1:1107:10684:14625': 0 -> 1
Below the original and newly created BAM files for the first read:
$ samtools view dedup.realigned.bam | grep
HWI-xxxxx:xxx:xxxxxxxxx:1:1106:17285:41358
HWI-xxxxx:xxx:xxxxxxxxx:1:1106:17285:41358 145 1 9932 0
74M 20 59443385 0
GGGGGGTGGGGGTCAGAGCACCACAGGGGGGGGGGGAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAAACC
########################################################=>=;?<<>=@?>1+)2);
BD:Z:MMMMSRQPPOOOONNNMMMMMMMMMMMMNNNNNNMMMMMMLLLLKKKKKKKKKKKKJJJJKKKKKKKMKFNNMM
PG:Z:MarkDuplicates RG:Z:some-sample1
BI:Z:FFFFCA@@AAAABCDEFGHIJJKKLLLLLLLLLMMMMMNNNNMMMMMMMLLLLLLLKKKKKKKKKKKLLHHGFF
NM:i:15 MQ:i:96 PQ:i:187 UQ:i:38 XQ:i:0
$ samtools view dedup.realigned.calmd.bam | grep
HWI-xxxxx:xxx:xxxxxxxxx:1:1106:17285:41358
HWI-xxxxx:xxx:xxxxxxxxx:1:1106:17285:41358 145 1 9932 0
74M 20 59443385 0
GGGGGGTGGGGGTCAGAGCACCACAGGGGGGGGGGGAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAAACC
########################################################)))))))))))))))*)*
BD:Z:MMMMSRQPPOOOONNNMMMMMMMMMMMMNNNNNNMMMMMMLLLLKKKKKKKKKKKKJJJJKKKKKKKMKFNNMM
PG:Z:MarkDuplicates RG:Z:some-sample1
BI:Z:FFFFCA@@AAAABCDEFGHIJJKKLLLLLLLLLMMMMMNNNNMMMMMMMLLLLLLLKKKKKKKKKKKLLHHGFF
MQ:i:96 PQ:i:187 UQ:i:38 XQ:i:0
ZQ:Z:@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@TUTRVSSUTWVUHB@H@Q
NM:i:70
MD:Z:0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0T4
My questions are:
Is the message 'different NM for read' because I picked up a wrong reference
file than the one used initially by bwa aligner?
In my eyes
MD:Z:0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0N0T4
means that now the reference contains N's while it did not have them in the
past.
If the @MD tag is correct it means the read should not be considered as
mapped to the chromosome 1 at all, and theoretically could have a better match
elsewhere. So, keeping it badly aligned in this locus is wrong I think. So
should I try to re-align all reads mentioned on the 'different NM for read'
line again? How? Remove them from BAM, mapping newly while creating a new BAM
file, then merging the BAM files? Sound I would loose some of the optional
columns so the merged BAM file would not have all columns for all reads. Is
that a problem?
Thank you,
Martin
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