TB-Profiler v6.7.0 fails with --caller bcftools (KeyError: invalid FORMAT: AD)

[paumayell]

Hi Jody,

I recently installed the latest version (v6.7.0) and encountered a consistent error when running TB-Profiler with --caller bcftools.

To troubleshoot, I tested with the provided test reads:
• Using --caller freebayes, the pipeline runs successfully.
• Using --caller bcftools on the same test reads, I get the same error observed with my own dataset.

The error appears to be related to the AD format field:
KeyError: 'invalid FORMAT: AD'

I’ve included additional details in the attached file.
error-v6.7.0-bcftools.txt

Could you please advise on how to proceed or if this might be a known issue?

Thanks in advance!

[paumayell]

Hey!
Just a quick update on this issue.
I reran the analysis using --caller bcftools but removed the --snp_dist 12 flag, and the pipeline completed successfully with my dataset. This suggests the error may be associated with the --snp_dist module/option rather than with bcftools itself.

[NabilaIsmail]

Hello! Thank you, removing the --snp_dist 12 flag fixed the issue, but now how will I be able to get the transmission graphs without this flag?

[jodyphelan]

Hi @paumayell and @NabilaIsmail apologies for the delay in getting back to you. I'll look into this now and get back to you asap!

[paumayell]

Hi Nabila and Jody, Thank you for your response. I did some additional testing and found that if I remove the --snp_dist 12 flag, the pipeline runs smoothly. However, in that case it does not generate the transmission graph (.json) output. If I use the flag, the pipeline stops before even generating the output files for bam/ and vcf/ directories. One question came up while testing: if I want to use --snp_dist 12, should I explicitly include the --call_whole_genomeflag, or is whole-genome calling automatically performed when --snp_dist is specified? I am very interested in using tb-profiler to calculate genetic distances and analyze relatedness within datasets. The tool already does a great job assigning lineages and drug resistance variants, so having a robust relatedness/transmission analysis would be a big plus. I am currently processing a large set of *Mycobacterium bovis* genomes, and tb-profiler has been one of the best tools I have found for this purpose. I am also interested in applying this pipeline to Nanopore-derived sequences (both *M. tuberculosis* and *M. bovis*). For relatedness analysis, I think it may be important to mask some genomic regions (for example repetitive regions, epitopes, and drug resistance-associated loci), similar to what MTBseq does. If tb-profiler could incorporate this kind of filtering, and maybe also generate an alignment for downstream phylogenetic analysis, it would make it a very complete pipeline for MTBC analysis, in my humble opinion :) In any case, thank you for your time, and for developing and maintaining such a useful tool. I really appreciate your work, and I hope my questions/suggestions are helpful. Best regards, Dra. Paulina M. Mejía Ponce Postdoctoral Researcher Escuela de Ciencias de la Salud Universidad Autónoma de Baja California Ensenada, BC, México

…

On Mon, May 4, 2026 at 1:26 AM Nabila Ismail ***@***.***> wrote: *NabilaIsmail* left a comment (jodyphelan/TBProfiler#479) <#479 (comment)> Hello, if you remove the --snp_dist 12 flag, do you still get the transmission graph in .json format? — Reply to this email directly, view it on GitHub <#479 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AE6R3RETJRBZJ5NZWE6ZYQD4ZBA4DAVCNFSM6AAAAACYDBMFXKVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHM2DGNRZGA2DAMZUGE> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[jodyphelan]

Ok I think I fixed the issue now. I'll push out a release for this week.

To answer some of your other questions:

One question came up while testing: if I want to use --snp_dist 12, should
I explicitly include the --call_whole_genomeflag, or is whole-genome
calling automatically performed when --snp_dist is specified?

No you can just do --sno_dist 12 and the --call_whole_genome flag will automatically be set.

I am also interested in applying this pipeline to Nanopore-derived
sequences (both M. tuberculosis and M. bovis). For relatedness
analysis, I think it may be important to mask some genomic regions (for
example repetitive regions, epitopes, and drug resistance-associated loci),
similar to what MTBseq does. If tb-profiler could incorporate this kind of
filtering, and maybe also generate an alignment for downstream phylogenetic
analysis, it would make it a very complete pipeline for MTBC analysis, in
my humble opinion :)

Yes 100% agree. At the moment these regions are masked out: https://github.com/jodyphelan/tbdb/blob/master/mask.bed
This is a combination of masks dericed from https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000465 and https://academic.oup.com/bioinformatics/article/38/7/1781/6502279?login=false