Biowulf High Performance Computing at the NIH
snippy: rapid haploid variant calling and core genome alignment

snippy is a tool for rapid haploid variant calling and core genome alignment.

Documentation
Important Notes

Interactive job
Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --mem=16g --gres=lscratch:10
[user@cn3200 ~]$module load snippy  
[+] Loading snippy 4.4.1  ...
Here is how snippy can be run on a test example:
[user@cn3200 ~]$ cp $SNIPPY_DATA/* .
[user@cn3200 ~]$ ln -s /fdb/igenomes/Homo_sapiens/UCSC/hg19/hg19.fa
[user@cn3200 ~]$ snippy --cpus 16 --outdir mysnps --ref hg19.fa  --R1  test_R1.fastq.gz --R2 test_R2.fastq.gz
...
[11:14:32] Written by Torsten Seemann 
[11:14:32] Obtained from https://github.com/tseemann/snippy
[11:14:32] Detected operating system: linux
[11:14:32] Enabling bundled linux tools.
[11:14:32] Found bwa - /opt/conda/envs/snippy/bin/bwa
[11:14:32] Found samtools - /opt/conda/envs/snippy/bin/samtools
[11:14:32] Found tabix - /opt/conda/envs/snippy/bin/tabix
[11:14:32] Found bgzip - /opt/conda/envs/snippy/bin/bgzip
[11:14:32] Found snpEff - /opt/conda/envs/snippy/bin/snpEff
[11:14:32] Found java - /opt/conda/envs/snippy/bin/java
[11:14:32] Found gzip - /usr/bin/gzip
[11:14:32] Found parallel - /opt/conda/envs/snippy/bin/parallel
[11:14:32] Found freebayes - /opt/conda/envs/snippy/bin/freebayes
[11:14:32] Found freebayes-parallel - /opt/conda/envs/snippy/bin/freebayes-parallel
[11:14:32] Found fasta_generate_regions.py - /opt/conda/envs/snippy/bin/fasta_generate_regions.py
[11:14:32] Found vcfstreamsort - /opt/conda/envs/snippy/bin/vcfstreamsort
[11:14:32] Found vcfuniq - /opt/conda/envs/snippy/bin/vcfuniq
[11:14:32] Found vcffirstheader - /opt/conda/envs/snippy/bin/vcffirstheader
[11:14:32] Found vcf-consensus - /opt/conda/envs/snippy/bin/../binaries/noarch/vcf-consensus
[11:14:32] Found snippy-vcf_to_tab - /opt/conda/envs/snippy/bin/snippy-vcf_to_tab
[11:14:32] Found snippy-vcf_report - /opt/conda/envs/snippy/bin/snippy-vcf_report
[11:14:32] Found snippy-vcf_filter - /opt/conda/envs/snippy/bin/snippy-vcf_filter
[11:14:32] Checking version: samtools --version is >= 1.3 - ok, have 1.6
[11:14:32] Checking version: freebayes --version is >= 1.1 - ok, have 1.3
[11:14:32] Checking version: snpEff -version is >= 4.3 - ok, have 5.1
[11:14:32] Using reference: /gpfs/gsfs7/users/user/snippy/hg19.fa
[11:14:50] Treating reference as 'fasta' format.
[11:14:50] Will use 16 CPU cores.
[11:14:50] Using read file: /gpfs/gsfs7/users/user/snippy/test_R1.fastq.gz
[11:14:50] Using read file: /gpfs/gsfs7/users/user/snippy/test_R2.fastq.gz
[11:14:50] Output folder mysnps3 already exists.
[user@cn1113 snippy]$ snippy --cpus 16 --outdir mysnps4 --ref hg19.fa  --R1  test_R1.fastq.gz --R2 test_R2.fastq.gz
[11:15:07] This is snippy 3.2-dev
[11:15:07] Written by Torsten Seemann 
[11:15:07] Obtained from https://github.com/tseemann/snippy
[11:15:07] Detected operating system: linux
[11:15:07] Enabling bundled linux tools.
[11:15:07] Found bwa - /opt/conda/envs/snippy/bin/bwa
[11:15:07] Found samtools - /opt/conda/envs/snippy/bin/samtools
[11:15:07] Found tabix - /opt/conda/envs/snippy/bin/tabix
[11:15:07] Found bgzip - /opt/conda/envs/snippy/bin/bgzip
[11:15:07] Found snpEff - /opt/conda/envs/snippy/bin/snpEff
[11:15:07] Found java - /opt/conda/envs/snippy/bin/java
[11:15:07] Found gzip - /usr/bin/gzip
[11:15:07] Found parallel - /opt/conda/envs/snippy/bin/parallel
[11:15:07] Found freebayes - /opt/conda/envs/snippy/bin/freebayes
[11:15:07] Found freebayes-parallel - /opt/conda/envs/snippy/bin/freebayes-parallel
[11:15:07] Found fasta_generate_regions.py - /opt/conda/envs/snippy/bin/fasta_generate_regions.py
[11:15:07] Found vcfstreamsort - /opt/conda/envs/snippy/bin/vcfstreamsort
[11:15:07] Found vcfuniq - /opt/conda/envs/snippy/bin/vcfuniq
[11:15:07] Found vcffirstheader - /opt/conda/envs/snippy/bin/vcffirstheader
[11:15:07] Found vcf-consensus - /opt/conda/envs/snippy/bin/../binaries/noarch/vcf-consensus
[11:15:07] Found snippy-vcf_to_tab - /opt/conda/envs/snippy/bin/snippy-vcf_to_tab
[11:15:07] Found snippy-vcf_report - /opt/conda/envs/snippy/bin/snippy-vcf_report
[11:15:07] Found snippy-vcf_filter - /opt/conda/envs/snippy/bin/snippy-vcf_filter
[11:15:07] Checking version: samtools --version is >= 1.3 - ok, have 1.6
[11:15:07] Checking version: freebayes --version is >= 1.1 - ok, have 1.3
[11:15:08] Checking version: snpEff -version is >= 4.3 - ok, have 5.1
[11:15:08] Using reference: /gpfs/gsfs7/users/user/snippy/hg19.fa
[11:15:19] Treating reference as 'fasta' format.
[11:15:19] Will use 16 CPU cores.
[11:15:19] Using read file: /gpfs/gsfs7/users/user/snippy/test_R1.fastq.gz
[11:15:19] Using read file: /gpfs/gsfs7/users/user/snippy/test_R2.fastq.gz
[11:15:19] Creating folder: mysnps4
[11:15:19] Changing working directory: mysnps4
[11:15:19] Creating reference folder: reference
[11:15:19] Extracting FASTA and GFF from reference.
[11:16:01] Wrote 25 sequences to ref.fa
[11:16:01] Wrote 0 features to ref.gff
[11:16:01] Freebayes will process 64 chunks of 49176391 bp, 16 chunks at a time.
[11:16:01] Using BAM RG (Read Group) ID: snps
[11:16:01] Running: samtools faidx reference/ref.fa 2>> snps.log
[11:16:17] Running: bwa index reference/ref.fa 2>> snps.log

[12:01:52] Running: mkdir reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa 2>> snps.log
[12:01:54] Running: mkdir reference/ref && bgzip -c reference/ref.gff > reference/ref/genes.gff.gz 2>> snps.log
[12:01:54] Running: (bwa mem  -v 2 -M -R '@RG\tID:snps\tSM:snps' -t 16 reference/ref.fa /gpfs/gsfs7/users/user/snippy/test_R1.fastq.gz /gpfs/gsfs7/users/user/snippy/test_R2.fastq.gz | samtools view -u -T reference/ref.fa -F 3844 -q 60 | samtools sort --reference reference/ref.fa > snps.bam) 2>> snps.log
[12:01:57] Running: samtools index snps.bam 2>> snps.log
[12:01:58] Running: samtools depth -aa -q 20 snps.bam | bgzip > snps.depth.gz 2>> snps.log
[12:07:08] Running: tabix -s 1 -b 2 -e 2 snps.depth.gz 2>> snps.log
[12:11:06] Running: fasta_generate_regions.py reference/ref.fa.fai 49176391 > reference/ref.txt 2>> snps.log
[12:11:07] Running: freebayes-parallel reference/ref.txt 16 -p 1 -q 20 -m 60 --min-coverage 10 -V -f reference/ref.fa snps.bam > snps.raw.vcf 2>> snps.log
[12:11:25] Running: /opt/conda/envs/snippy/bin/snippy-vcf_filter --minqual 10 --mincov 10 --minfrac 0.9  snps.raw.vcf > snps.filt.vcf 2>> snps.log
[12:11:25] Running: cp snps.filt.vcf snps.vcf 2>> snps.log
[12:11:25] Running: bgzip -c snps.vcf > snps.vcf.gz 2>> snps.log
[12:11:25] Running: tabix -p vcf snps.vcf.gz 2>> snps.log
[12:11:25] Running: /opt/conda/envs/snippy/bin/snippy-vcf_to_tab --gff reference/ref.gff --ref reference/ref.fa --vcf snps.vcf > snps.tab 2>> snps.log
[12:11:49] Running: vcf-consensus snps.vcf.gz < reference/ref.fa > snps.consensus.fa 2>> snps.log
[12:13:43] Running: /opt/conda/envs/snippy/bin/snippy-vcf_filter --subs snps.filt.vcf > snps.filt.subs.vcf 2>> snps.log
[12:13:43] Running: bgzip -c snps.filt.subs.vcf > snps.filt.subs.vcf.gz 2>> snps.log
[12:13:43] Running: tabix -p vcf snps.filt.subs.vcf.gz 2>> snps.log
[12:13:43] Running: vcf-consensus snps.filt.subs.vcf.gz < reference/ref.fa > snps.consensus.subs.fa 2>> snps.log
[12:15:40] Generating aligned/masked FASTA relative to reference: snps.aligned.fa

...

[12:46:32] Creating extra output files: BED GFF CSV TXT HTML
[12:46:32] Identified 3 variants.
[12:46:32] Result folder: mysnps4
[12:46:32] Result files:
[12:46:32] * mysnps4/snps.aligned.fa
[12:46:32] * mysnps4/snps.bam
[12:46:32] * mysnps4/snps.bam.bai
[12:46:32] * mysnps4/snps.bed
[12:46:32] * mysnps4/snps.consensus.fa
[12:46:32] * mysnps4/snps.consensus.subs.fa
[12:46:32] * mysnps4/snps.csv
[12:46:32] * mysnps4/snps.depth.gz
[12:46:32] * mysnps4/snps.depth.gz.tbi
[12:46:32] * mysnps4/snps.filt.subs.vcf
[12:46:32] * mysnps4/snps.filt.subs.vcf.gz
[12:46:32] * mysnps4/snps.filt.subs.vcf.gz.tbi
[12:46:32] * mysnps4/snps.filt.vcf
[12:46:32] * mysnps4/snps.gff
[12:46:32] * mysnps4/snps.html
[12:46:32] * mysnps4/snps.log
[12:46:32] * mysnps4/snps.raw.vcf
[12:46:32] * mysnps4/snps.tab
[12:46:32] * mysnps4/snps.txt
[12:46:32] * mysnps4/snps.vcf
[12:46:32] * mysnps4/snps.vcf.gz
[12:46:32] * mysnps4/snps.vcf.gz.tbi
[12:46:32] Walltime used: 1 hours, 31 minutes, 25 seconds
[12:46:32] Found a bug? Post it at https://github.com/tseemann/snippy/issues
[12:46:32] Done.
An output folder mysnps is created:
[user@cn3200 ~]$ tree mysnps
mysnps
├ reference
│   ├ genomes
│   │   └ ref.fa
│   ├ ref
│   │   ├ genes.gff.gz
│   │   └ snpEffectPredictor.bin
│   ├ ref.fa
│   ├ ref.fa.amb
│   ├ ref.fa.ann
│   ├ ref.fa.bwt
│   ├ ref.fa.fai
│   ├ ref.fa.pac
│   ├ ref.fa.sa
│   ├ ref.gff
│   ├ ref.txt
│   └ snpeff.config
├ ref.fa -> reference/ref.fa
├ ref.fa.fai -> reference/ref.fa.fai
├ snps.aligned.fa
├ snps.bam
├ snps.bam.bai
├ snps.bed
├ snps.consensus.fa
├ snps.consensus.subs.fa
├ snps.csv
├ snps.filt.vcf
├ snps.gff
├ snps.html
├ snps.log
├ snps.raw.vcf
├ snps.subs.vcf
├ snps.tab
├ snps.txt
├ snps.vcf
├ snps.vcf.gz
└ snps.vcf.gz.csi

3 directories, 33 filesa
End the interactive session:
[user@cn3200 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$