#Snippy Rapid haploid variant calling and core SNP phylogeny
##Author Torsten Seemann (@torstenseemann)
##Synopsis
Snippy finds SNPs between a haploid reference genome and your NGS sequence reads. It will find both substitutions (snps) and insertions/deletions (indels). It will use as many CPUs as you can give it on a single computer (tested to 64 cores). It is designed with speed in mind, and produces a consistent set of output files in a single folder. It can then take a set of Snippy results using the same reference and generate a core SNP alignment (and ultimately a phylogenomic tree).
##Quick Start
% snippy --cpus 16 --outdir mysnps --ref Listeria.gbk --R1 FDA_R1.fastq.gz --R1 FDA_R2.fastq.gz
<cut>
Walltime used: 3 min, 42 sec
Results folder: mysnps
Done.
% ls mysnps
snps.vcf snps.bed snps.gff snps.csv snps.tab snps.html
snps.bam snps.txt reference/ ...
% head -5 mysnps/snps.tab
CHROM POS TYPE REF ALT EVIDENCE FTYPE STRAND NT_POS AA_POS LOCUS_TAG GENE PRODUCT EFFECT
chr 5958 snp A G G:44 A:0 CDS + 41/600 13/200 ECO_0001 dnaA replication protein DnaA missense_variant c.548A>C p.Lys183Thr
chr 35524 snp G T T:73 G:1 C:1 tRNA -
chr 45722 ins ATT ATTT ATTT:43 ATT:1 CDS - ECO_0045 gyrA DNA gyrase
chr 100541 del CAAA CAA CAA:38 CAAA:1 CDS + ECO_0179 hypothetical protein
plas 619 complex GATC AATA GATC:28 AATA:0
plas 3221 mnp GA CT CT:39 CT:0 CDS + ECO_p012 rep hypothetical protein
% snippy-core --prefix core mysnps1 mysnps2 mysnps3 mysnps4
Loaded 4 SNP tables.
Found 2814 core SNPs from 96615 SNPs.
% ls core.*
core.aln core.tab core.txt
#Installation
##Homebrew Install HomeBrew (Mac OS X) or LinuxBrew (Linux).
brew tap homebrew/science
brew tap chapmanb/cbl
brew tap tseemann/homebrew-bioinformatics-linux
brew install snippy
snippy --help
##Source
This will install the latest version direct from Github. You'll need to add the bin directory to your PATH.
cd $HOME
git clone https://github.com/tseemann/snippy.git
$HOME/snippy/bin/snippy --help
#Calling SNPs
##Input Requirements
- a reference genome in FASTA or GENBANK format (can be in multiple contigs)
- sequence read files in FASTQ or FASTA format (can be .gz compressed) format
- a folder to put the results in
##Output Files
| Extension | Description |
|---|---|
| .tab | A simple tab-separated summary of all the variants |
| .csv | A comma-separated version of the .tab file |
| .html | A HTML version of the .tab file |
| .vcf | The final annotated variants in VCF format |
| .vcf.gz | Compressed .vcf file via BGZIP |
| .vcf.gz.tbi | Index for the .vcf.gz via TABIX |
| .bed | The variants in BED format |
| .gff | The variants in GFF3 format |
| .bam | The alignments in BAM format. Note that multi-mapping and unmapped reads are not present. |
| .bam.bai | Index for the .bam file |
| .raw.vcf | The unfiltered variant calls from Freebayes |
| .filt.vcf | The filtered variant calls from Freebayes |
| .log | A log file with the commands run and their outputs |
| .consensus.fa | A version of the reference genome with all variants instantiated |
| .aligned.fa | A version of the reference but with - for unaligned and N for depth < --minfrac (does not have variants) |
| .depth.gz | Output of samtools depth for the .bam file |
| .depth.gz.tbi | Index for the .depth.gz (currently unused) |
##Columns in the TAB/CSV/HTML formats
| Name | Description |
|---|---|
| CHROM | The sequence the variant was found in eg. the name after the > in the FASTA reference |
| POS | Position in the sequence, counting from 1 |
| TYPE | The variant type: snp msp ins del complex |
| REF | The nucleotide(s) in the reference |
| ALT | The alternate nucleotide(s) supported by the reads |
| EVIDENCE | Frequency counts for REF and ALT |
If you supply a Genbank file as the --reference rather than a FASTA file, Snippy will fill in these extra columns by using the genome annotation to tell you which feature was affected by the variant:
| Name | Description |
|---|---|
| FTYPE | Class of feature affected: CDS tRNA rRNA ... |
| STRAND | Strand the feature was on: + - . |
| NT_POS | Nucleotide position of the variant withinthe feature / Length in nt |
| AA_POS | Residue position / Length in aa (only if FTYPE is CDS) |
| LOCUS_TAG | The /locus_tag of the feature (if it existed) |
| GENE | The /gene tag of the feature (if it existed) |
| PRODUCT | The /product tag of the feature (if it existed) |
| EFFECT | The snpEff annotated consequence of this variant |
##Variant Types
| Type | Name | Example |
|---|---|---|
| snp | Single Nucleotide Polymorphism | A => T |
| mnp | Multiple Nuclotide Polymorphism | GC => AT |
| ins | Insertion | ATT => AGTT |
| del | Deletion | ACGG => ACG |
| complex | Combination of snp/mnp | ATTC => GTTA |
##The variant caller The variant calling is done by Freebayes. However, Snippy uses a very simple model for reporting variants, relying on two main options:
--mincovis the minimum number of reads covering the variant position.--minfracis the minimum proportion of those reads which must differ from the reference.
By default Snippy uses --mincov 10 --minfrac 0.9 which is reasonable for most cases, but for very high coverage data you may get mixed populations such as (REF:310 ALT:28). Snippy may use a more statistical approach in future versions like Nesoni does.
#Core SNP phylogeny
If you call SNPs for multiple isolates from the same reference, you can produce an alignment of "core SNPs" which can be used to build a high-resolution phylogeny (ignoring possible recombination). A "core site" is a genomic position that is present in all the samples. A core site can have the same nucleotide in every sample ("monomorphic") or some samples can be different ("polymorphic" or "variant"). If we ignore the complications of "ins", "del" variant types, and just use variant sites, these are the "core SNP genome".
##Input Requirements
- a set of Snippy folders which used the same
--refsequence.
##Output Files
| Extension | Description |
|---|---|
| .aln | A core SNP alignment in the --aformat format (default FASTA) |
| .tab | Tab-separated columnar list of core SNP sites with alleles and annotations |
| .txt | Tab-separated columnar list of alignment/core-size statistics |
#Information
##Etymology The name Snippy is a combination of SNP (pronounced "snip") , snappy (meaning "quick") and Skippy the Bush Kangaroo (to represent its Australian origin)
##License Snippy is free software, released under the GPL (version 3).
##Issues Please submit suggestions and bug reports here: https://github.com/tseemann/snippy/issues
##Requirements
- Perl >= 5.6
- BioPerl >= 1.6
- bwa mem >= 0.7.12
- samtools >= 1.1
- GNU parallel > 2013xxxx
- freebayes >= 0.9.20
- freebayes sripts (freebayes-parallel, fasta_generate_regions.py)
- vcflib (vcffilter, vcfstreamsort, vcfuniq, vcffirstheader)
- vcftools (vcf-consensus)
- snpEff >= 4.1
##Bundled binaries For a modern Linux system (Ubuntu >= 12.04) and Mac OS X all the binaries, JARs and scripts are included.