empty Snippy output

[Mahjabeenkhan58]

Hi, I am running snippy but it is providing empty .tab and .csv file which means no output. I am attaching the log file so if anyone can help me. snps.log

echo snippy 4.6.0

cd /srv/scratch/z5045473/GP

/apps/snippy/4.6.0/bin/snippy --cpus 16 --outdir mysnps --ref /srv/scratch/z5045473/GP/isolate2/sequence.gb --R1 /srv/scratch/z5045473/GP/isolate2/UJ2_1.fastq.gz --R2 /srv/scratch/z5045473/GP/isolate2/UJ2_2.fastq.gz

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA... 0.02 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 1.05 seconds elapse. [bwa_index] Update BWT... 0.02 sec [bwa_index] Pack forward-only FASTA... 0.03 sec [bwa_index] Construct SA from BWT and Occ... 0.50 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index reference/ref.fa [main] Real time: 1.759 sec; CPU: 1.627 sec

mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

ln -sf reference/ref.fa .

ln -sf reference/ref.fa.fai .

mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz

snpEff build -c reference/snpeff.config -dataDir . -gff3 ref

WARNING: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate.

bwa mem -Y -M -R '@RG\tID:mysnps\tSM:mysnps' -t 16 reference/ref.fa /srv/scratch/z5045473/GP/isolate2/UJ2_1.fastq.gz /srv/scratch/z5045473/GP/isolate2/UJ2_2.fastq.gz | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /scratch/pbs.2803002.kman.restech.unsw.edu.au --threads 7 -m 1000M | samtools fixmate -m --threads 7 - - | samtools sort -l 0 -T /scratch/pbs.2803002.kman.restech.unsw.edu.au --threads 7 -m 1000M | samtools markdup -T /scratch/pbs.2803002.kman.restech.unsw.edu.au --threads 7 -r -s - - > snps.bam

READ 18098514 WRITTEN 18077493 EXCLUDED 18074177 EXAMINED 24337 PAIRED 1690 SINGLE 22647 DULPICATE PAIR 282 DUPLICATE SINGLE 20739 DUPLICATE TOTAL 21021

samtools index snps.bam

fasta_generate_regions.py reference/ref.fa.fai 172938 > reference/ref.txt

freebayes-parallel reference/ref.txt 16 -p 2 -P 0 -C 2 -F 0.05 --min-coverage 10 --min-repeat-entropy 1.0 -q 13 -m 60 --strict-vcf -f reference/ref.fa snps.bam > snps.raw.vcf

bcftools view --include 'FMT/GT="1/1" && QUAL>=100 && FMT/DP>=10 && (FMT/AO)/(FMT/DP)>=0' snps.raw.vcf | vt normalize -r reference/ref.fa - | bcftools annotate --remove '^INFO/TYPE,^INFO/DP,^INFO/RO,^INFO/AO,^INFO/AB,^FORMAT/GT,^FORMAT/DP,^FORMAT/RO,^FORMAT/AO,^FORMAT/QR,^FORMAT/QA,^FORMAT/GL' > snps.filt.vcf

snpEff ann -noLog -noStats -no-downstream -no-upstream -no-utr -c reference/snpeff.config -dataDir . ref snps.filt.vcf > snps.vcf

/apps/snippy/4.6.0/bin/snippy-vcf_to_tab --gff reference/ref.gff --ref reference/ref.fa --vcf snps.vcf > snps.tab

Loading reference: reference/ref.fa Loaded 1 sequences. Loading features: reference/ref.gff Parsing variants: snps.vcf Converted 0 SNPs to TAB format.

/apps/snippy/4.6.0/bin/snippy-vcf_extract_subs snps.filt.vcf > snps.subs.vcf

bcftools convert -Oz -o snps.vcf.gz snps.vcf

bcftools index -f snps.vcf.gz

bcftools consensus --sample mysnps -f reference/ref.fa -o snps.consensus.fa snps.vcf.gz

bcftools convert -Oz -o snps.subs.vcf.gz snps.subs.vcf

bcftools index -f snps.subs.vcf.gz

bcftools consensus --sample mysnps -f reference/ref.fa -o snps.consensus.subs.fa snps.subs.vcf.gz

rm -f snps.subs.vcf.gz snps.subs.vcf.gz.csi snps.subs.vcf.gz.tbi

[Mahjabeenkhan58]

@tseemann Please if you can see this

[jesuscamara]

Same here, I have tried to troubleshoot this by checking the previous issues related to the subject, but no luck.

[amanzanom]

I am having the same problem when running snippy. Have tried the embl file, genbank, and fasta with the same results. Same data as input for breseq is working. I can provide run and log files if needed to debug this. It would be great if this can be fixed, as I could find the problem reported all over the place, but no solution.

[alex-trist]

Same here. No SNP's written in output. Here is my log

echo snippy 4.6.0

cd /home/microseq/Seq_HUMS/VRE3

/home/microseq/anaconda3/envs/snippy2/bin/snippy --cpus 22 --cleanup --outdir snippy/349333 --reference Ref_VRE_VanA_ST612.fasta --R1 trimm/349333_R1_trim.fastq.gz --R2 trimm/349333_R2_trim.fastq.gz

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA... 0.01 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 0.30 seconds elapse. [bwa_index] Update BWT... 0.01 sec [bwa_index] Pack forward-only FASTA... 0.00 sec [bwa_index] Construct SA from BWT and Occ... 0.11 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index reference/ref.fa [main] Real time: 0.456 sec; CPU: 0.427 sec

mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

ln -sf reference/ref.fa .

ln -sf reference/ref.fa.fai .

mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz

bwa mem -Y -M -R '@RG\tID:349333\tSM:349333' -t 22 reference/ref.fa /home/microseq/Seq_HUMS/VRE3/trimm/349333_R1_trim.fastq.gz /home/microseq/Seq_HUMS/VRE3/trimm/349333_R2_trim.fastq.gz | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /tmp --threads 10 -m 727M | samtools fixmate -m --threads 10 - - | samtools sort -l 0 -T /tmp --threads 10 -m 727M | samtools markdup -T /tmp --threads 10 -r -s - - > snps.bam

COMMAND: samtools markdup -T /tmp --threads 10 -r -s - - READ: 7443387 WRITTEN: 6544423 EXCLUDED: 1472010 EXAMINED: 5971377 PAIRED: 5741992 SINGLE: 229385 DUPLICATE PAIR: 690176 DUPLICATE SINGLE: 208788 DUPLICATE PAIR OPTICAL: 0 DUPLICATE SINGLE OPTICAL: 0 DUPLICATE NON PRIMARY: 0 DUPLICATE NON PRIMARY OPTICAL: 0 DUPLICATE PRIMARY TOTAL: 898964 DUPLICATE TOTAL: 898964 ESTIMATED_LIBRARY_SIZE: 10965269

samtools index snps.bam

fasta_generate_regions.py reference/ref.fa.fai 67912 > reference/ref.txt

freebayes-parallel reference/ref.txt 22 -p 2 -P 0 -C 2 -F 0.05 --min-coverage 10 --min-repeat-entropy 1.0 -q 13 -m 60 --strict-vcf -f reference/ref.fa snps.bam > snps.raw.vcf

bcftools view --include 'FMT/GT="1/1" && QUAL>=100 && FMT/DP>=10 && (FMT/AO)/(FMT/DP)>=0' snps.raw.vcf | vt normalize -r reference/ref.fa - | bcftools annotate --remove '^INFO/TYPE,^INFO/DP,^INFO/RO,^INFO/AO,^INFO/AB,^FORMAT/GT,^FORMAT/DP,^FORMAT/RO,^FORMAT/AO,^FORMAT/QR,^FORMAT/QA,^FORMAT/GL' > snps.filt.vcf

cp snps.filt.vcf snps.vcf

/home/microseq/anaconda3/envs/snippy2/bin/snippy-vcf_to_tab --gff reference/ref.gff --ref reference/ref.fa --vcf snps.vcf > snps.tab

Loading reference: reference/ref.fa Loaded 191 sequences. Loading features: reference/ref.gff Parsing variants: snps.vcf Converted 0 SNPs to TAB format.

/home/microseq/anaconda3/envs/snippy2/bin/snippy-vcf_extract_subs snps.filt.vcf > snps.subs.vcf

bcftools convert -Oz -o snps.vcf.gz snps.vcf

bcftools index -f snps.vcf.gz

bcftools consensus --sample 349333 -f reference/ref.fa -o snps.consensus.fa snps.vcf.gz

Applied 0 variants

bcftools convert -Oz -o snps.subs.vcf.gz snps.subs.vcf

bcftools index -f snps.subs.vcf.gz

bcftools consensus --sample 349333 -f reference/ref.fa -o snps.consensus.subs.fa snps.subs.vcf.gz

Applied 0 variants

rm -f snps.subs.vcf.gz snps.subs.vcf.gz.csi snps.subs.vcf.gz.tbi

Any help will be much appreciated!

Thanks.