Error in running snippy-multi

[adimascf]

I get an error message when running snippy-multi for my .fastq files ERROR: Could not find .aligned.fa/.vcf in (snippy folder). When I open the folder indeed .aligned.fa/.vcf is not found. The snps.log file says [markdup] error reading header. The .bam file is empty as well.
I am trying using contigs as input but getting the same error. Is there any solution to this issue?
Thank you

[alanxelena]

Jumping in with an unsolicited opinion. Could it be that some of the dependencies are not working? try doing snippy --check

[adimascf]

Thank you for the reply @alanxelena

Yes, you are correct, that is the dependencies issue. I need to have samtools version >= 1.3. I tried to run snippy check, and get this output:

[15:56:47] This is snippy 3.2-dev
[15:56:47] Written by Torsten Seemann <[email protected]>
[15:56:47] Obtained from https://github.com/tseemann/snippy
[15:56:47] Detected operating system: linux
[15:56:47] Enabling bundled linux tools.
[15:56:47] Found bwa - /home/adimascf/miniconda3/envs/snippy-env/bin/bwa
[15:56:47] Found samtools - /home/adimascf/miniconda3/envs/snippy-env/bin/samtools
[15:56:47] Found tabix - /home/adimascf/miniconda3/envs/snippy-env/bin/tabix
[15:56:47] Found bgzip - /home/adimascf/miniconda3/envs/snippy-env/bin/bgzip
[15:56:47] Found snpEff - /home/adimascf/miniconda3/envs/snippy-env/bin/snpEff
[15:56:47] Found java - /home/adimascf/miniconda3/envs/snippy-env/bin/java
[15:56:47] Found gzip - /usr/bin/gzip
[15:56:47] Found parallel - /home/adimascf/miniconda3/envs/snippy-env/bin/parallel
[15:56:47] Found freebayes - /home/adimascf/miniconda3/envs/snippy-env/bin/freebayes
[15:56:47] Found freebayes-parallel - /home/adimascf/miniconda3/envs/snippy-env/bin/freebayes-parallel
[15:56:47] Found fasta_generate_regions.py - /home/adimascf/miniconda3/envs/snippy-env/bin/fasta_generate_regions.py
[15:56:47] Found vcfstreamsort - /home/adimascf/miniconda3/envs/snippy-env/bin/vcfstreamsort
[15:56:47] Found vcfuniq - /home/adimascf/miniconda3/envs/snippy-env/bin/vcfuniq
[15:56:47] Found vcffirstheader - /home/adimascf/miniconda3/envs/snippy-env/bin/vcffirstheader
[15:56:47] Found vcf-consensus - /usr/local/bin/vcf-consensus
[15:56:47] Found snippy-vcf_to_tab - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_to_tab
[15:56:47] Found snippy-vcf_report - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_report
[15:56:47] Found snippy-vcf_filter - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_filter
[15:56:47] Need samtools --version >= 1.3 - please upgrade it.

But, it is a weird, beacuse I get this output when I run samtools --version:

samtools 1.16.1
Using htslib 1.16
Copyright (C) 2022 Genome Research Ltd.
.....

I have samtools version 1.16.1 installed in my computer.

[alanxelena]

Hey @adimascf

Happy I could help. There is an issue with the way snippy detects versions, so 1.16 is apparently read as 1.1 and so you get this error. There was a thread about it somewhere, I don't know if it was here or somewhere else, I'll have to check. The solution was to make a little modification in snippy's code.
I'll post the link to the solution whenever I find it.

Cheers!

EDIT: Here it is #344 The solution was posted by a user called chienchi

[adimascf]

Thank you again @alanxelena. I have downgraded the samtools version to 1.9 now, using conda install -c bioconda samtools=1.9 like in this #344 issue.

snippy check

[17:02:49] This is snippy 3.2-dev
[17:02:49] Written by Torsten Seemann <[email protected]>
[17:02:49] Obtained from https://github.com/tseemann/snippy
[17:02:49] Detected operating system: linux
[17:02:49] Enabling bundled linux tools.
[17:02:49] Found bwa - /home/adimascf/miniconda3/envs/snippy-env/bin/bwa
[17:02:49] Found samtools - /home/adimascf/miniconda3/envs/snippy-env/bin/samtools
[17:02:49] Found tabix - /home/adimascf/miniconda3/envs/snippy-env/bin/tabix
[17:02:49] Found bgzip - /home/adimascf/miniconda3/envs/snippy-env/bin/bgzip
[17:02:49] Found snpEff - /home/adimascf/miniconda3/envs/snippy-env/bin/snpEff
[17:02:49] Found java - /home/adimascf/miniconda3/envs/snippy-env/bin/java
[17:02:49] Found gzip - /usr/bin/gzip
[17:02:49] Found parallel - /home/adimascf/miniconda3/envs/snippy-env/bin/parallel
[17:02:49] Found freebayes - /home/adimascf/miniconda3/envs/snippy-env/bin/freebayes
[17:02:49] Found freebayes-parallel - /home/adimascf/miniconda3/envs/snippy-env/bin/freebayes-parallel
[17:02:49] Found fasta_generate_regions.py - /home/adimascf/miniconda3/envs/snippy-env/bin/fasta_generate_regions.py
[17:02:49] Found vcfstreamsort - /home/adimascf/miniconda3/envs/snippy-env/bin/vcfstreamsort
[17:02:49] Found vcfuniq - /home/adimascf/miniconda3/envs/snippy-env/bin/vcfuniq
[17:02:49] Found vcffirstheader - /home/adimascf/miniconda3/envs/snippy-env/bin/vcffirstheader
[17:02:49] Found vcf-consensus - /usr/local/bin/vcf-consensus
[17:02:49] Found snippy-vcf_to_tab - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_to_tab
[17:02:49] Found snippy-vcf_report - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_report
[17:02:49] Found snippy-vcf_filter - /home/adimascf/miniconda3/envs/snippy-env/bin/snippy-vcf_filter
[17:02:49] Checking version: samtools --version is >= 1.3 - ok, have 1.9
[17:02:49] Checking version: freebayes --version is >= 1.1 - ok, have 1.3
[17:02:50] Checking version: snpEff -version is >= 4.3 - ok, have 5.1
[17:02:50] Please supply a reference FASTA/GBK/EMBL file with --reference

Seems it will work now, I will try using my data later.
Thank you

[alanxelena]

Oh, yes, downgrading was another option, I don't know if there were many major changes that can affect the functionality of snippy though. Glad it works!