No MD tag found

[nirwan1265]

I am getting this error: [ntanduk@c050n01 Introgress]$ ./IntroMap.py -i PN2_SID100_bowtie2.bam -r chr1_reference_Zm_v5.fa -o PN2_SN100 -t 0.90 -b True -f 0.05 ERROR: No MD tag found! Please ensure that your aligner generated MD tags.

This is what i have and I do believe there is MD tags in them: (base) [ntanduk@login01 PN2_SID100]$ samtools view -h PN2_SID100_bowtie2.bam | more @HD VN:1.0 SO:coordinate @SQ SN:chr1 LN:308452471 ....... ATATTCAATAATACCGAAGGCTAACACGTACGGATTTTAGACCAAGAAATGGTCTCCACCAGAAATCCAAGAATGTGATCTATGGCAAGGAAACATATGT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF FFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFFFFFFF:FFFFFFFF:F:,FFFFFFFFFFFF:FFFFFF:FFF:FFFFF:FFFFFF AS:i:-55 XS:i:-49 XN:i:0 XM: i:11 XO:i:0 XG:i:0 NM:i:11 MD:Z:1C1A3A0A3C0C0A23C6T24C10T61 YS:i:-3 YT:Z:CP A00600:293:H7HYFDSX7:1:1513:21811:17910 99 chr1 11142 1 143M = 11397 398 CTGATAAATGGCCACACAACCCCCATTTTTTATGAAAATAGCC ATGAGCGACCATTTTCAATAATATTAGAGGCTAAGACCTACGGATTTTTGACCAAGAAATGGTCTCCACCAGAAATCCAAGAATGTGATCTATGGCAAGG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF FFFFFFFFFFFFFFFFF,FFFFF:,,:FFFFF:F,FF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-5 XS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG: i:0 NM:i:1 MD:Z:0A142 YS:i:0 YT:Z:CP A00600:293:H7HYFDSX7:2:1436:13630:27790 147 chr1 11294 2 85M = 10972 -407 TGGGGTTAGGTTTATGAGCCTCTGGTCGATGATCAATGGCCAC ACAACCCCCATTTTTGTCGAAAATAGCCATGAACGACCATTT F:FFF,:FFF:,FF,:FFF,FF,F,FFFFFF:FF:F:FF,F,FFF::FFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFF AS:i:-3 XS:

I have three 3 alignment software - bowtie2, minimap2, and bwa. I still get the same error. Any help is appreciated

[danshea]

Hi Nirwan,

You may have unaligned reads in the file. Have you tried filtering any unaligned reads prior to feeding it to IntroMap?

(i.e. - See https://www.biostars.org/p/56246/ on using samtools to confirm if there are any unmapped reads)

Regards, Dan

[nirwan1265]

Yes. I removed the unmapped region and did the picard deduplication marking and it worked. thanks!!