Could not find .aligned.fa/.vcf in 73

zhichusun commented 2 years ago

I'm trying to run snippy-multi for a few assembled genomes. 73 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/73.fasta 75 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/75.fasta 95 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/95.fasta A1 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/A1.fasta A6 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/A6.fasta D-3-11 /xulie/shiyanshijiyuanfenxi/jiyuanxulie/D-3-11.fasta snippy-multi input.tab --reference Reference.gbk > 1.sh ./1.sh

The final result. This is snippy-core 4.6.0 Obtained from http://github.com/tseemann/snippy Enabling bundled tools for linux Found any2fasta - /root/miniconda3/envs/snippy/bin/any2fasta Found samtools - /root/miniconda3/envs/snippy/bin/samtools Found minimap2 - /root/miniconda3/envs/snippy/bin/minimap2 Found bedtools - /root/miniconda3/envs/snippy/bin/bedtools Found snp-sites - /root/miniconda3/envs/snippy/bin/snp-sites Saving reference FASTA: core.ref.fa This is any2fasta 0.4.2 Opening '73/ref.fa' Detected FASTA format Read 27360 lines from '73/ref.fa' Wrote 1 sequences from FASTA file. Processed 1 files. Done. Loaded 1 sequences totalling 1641481 bp. Will mask 0 regions totalling 0 bp ~ 0.00% ERROR: Could not find .aligned.fa/.vcf in 73

Viewed the log results in each file showing WARNING_GENE_NOT_FOUND: Too many 'WARNING_GENE_NOT_FOUND' warnings, no further warnings will be shown. WARNING_FRAMES_ZERO: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate. ERROR: CDS check file '/xulie/shiyanshijiyuanfenxi/jiyuanxulie/73/reference/./ref/cds.fa' not found. ERROR: Protein check file '/xulie/shiyanshijiyuanfenxi/jiyuanxulie/73/reference/./ref/protein.fa' not found. ERROR: Database check failed.

I don't know what to do can you help me

caitiecollins commented 2 years ago

Dear @tseemann,

I am unfortunately running into a similar error. I'm trying to run snippy-multi on a set of assembled genomes. I did this with no issues when using my reference genome in fasta format. But I wanted to re-run snippy with the genbank version of the reference genome to retain the gene annotation info.

This failed with the same errors as @zhichusun. (Full snippy output dialogue attached.): WARNING_FRAMES_ZERO: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate. ERROR: CDS check file '/media/bitlockermount/Caitlin/Work/JP PHE/panaroo_data/pseudomonas/snippy/snippy2/21946_8#187_K11/reference/./ref/cds.fa' not found. ERROR: Protein check file '/media/bitlockermount/Caitlin/Work/JP PHE/panaroo_data/pseudomonas/snippy/snippy2/21946_8#187_K11/reference/./ref/protein.fa' not found. ERROR: Database check failed.

For some reason, snippy seemed unable to recognise the annotations in the genbank reference or to link these to the relevant sequences in the genomes. Each of the snps.log files contains repeated instances of: WARNING_TRANSCRIPT_NOT_FOUND, WARNING_GENE_NOT_FOUND.

I will attach the snps.log file for one of my genomes, as well as the genbank file for the reference genome (note: couldn't upload .gb to github; saved as .gb.txt).

Please let me know if there is anything wrong with the format of the genbank reference, or if something else is missing or incorect. Thank you for any suggestions.

snippy output - error w ref.gb.txt 21946_8#187_K11 - snps.log runme.sh.txt Pseudomonas_aeruginosa_PAO1_v2.gb.txt

liaochenlanruo commented 2 years ago

@zhichusun @caitiecollins You should check the version of snpEff, the error may happen with v5.1, and v5.0 is OK. I don't know why, but you can have a try.

zhichusun commented 2 years ago

I found that the default memory for the program to run is too large, and my virtual machine memory is not enough. The running code can be changed to this snippy --cpu 1 --outdir mut1 --ref 1.gb --ctgs 1.fasta --ram 1

jimie0311 commented 2 years ago

@zhichusun @caitiecollins You should check the version of snpEff, the error may happen with v5.1, and v5.0 is OK. I don't know why, but you can have a try.

Hi liaochenlanruo， I met the same error like them.

This is snippy-core 4.6.0 Obtained from http://github.com/tseemann/snippy Enabling bundled tools for linux Found any2fasta - /data/Liangjunmin/opt/biosoft/snippy/binaries/noarch/any2fasta Found samtools - /usr/bin/samtools Found minimap2 - /data/Liangjunmin/opt/biosoft/snippy/binaries/linux/minimap2 Found bedtools - /usr/bin/bedtools Found snp-sites - /data/Liangjunmin/opt/biosoft/snippy/binaries/linux/snp-sites Saving reference FASTA: core.ref.fa This is any2fasta 0.4.2 Opening 'GCA_000599445.1/ref.fa' Detected FASTA format Read 634061 lines from 'GCA_000599445.1/ref.fa' Wrote 5 sequences from FASTA file. Processed 1 files. Done. Loaded 5 sequences totalling 38043241 bp. Will mask 0 regions totalling 0 bp ~ 0.00% 0 GCA_000599445.1 snp=92950 del=9924 ins=8021 het=3091 unaligned=2289544 1 GCA_000966635.1 snp=79747 del=12531 ins=5687 het=1244 unaligned=2476255 2 GCA_000966645.1 snp=120306 del=16916 ins=9597 het=2187 unaligned=2672285 3 GCA_001717905.1 snp=120409 del=13623 ins=10844 het=4413 unaligned=2627887 4 GCA_001717915.1 snp=90738 del=11109 ins=8114 het=2954 unaligned=2305564 5 GCA_002352725.1 snp=122605 del=12274 ins=11062 het=4418 unaligned=2525283 ERROR: Could not find .aligned.fa/.vcf in GCA_006942295.1

My snpEff is version SnpEff 4.3t, Is it too old? I also changed the cpu to 1 as zhichusun suggested but it didn't work.

gcronan commented 2 years ago

@zhichusun @caitiecollins You should check the version of snpEff, the error may happen with v5.1, and v5.0 is OK. I don't know why, but you can have a try.

Downgrading enpEff from v5.1 to v5.0 fixed this error for me too.