ekidna

Assembly based core genome SNP alignments

Introduction

In an ideal world, to determine a core genome amongst a set of genomes, we would perform a "multiple whole genome alignment" and extract the conserved sites (mono- and poly- morphic). Software like Mauve can do this, but it does not scale to more than 10s of genomes, due to the exponential computational need.

Instead, we usually choose a reference genome and align isolate genomes sequentially to the reference. The "genomes" could be already assembled genomes (contigs in FASTA) or raw sequencing data (reads in FASTQ). Tools like ParSNP and Roary can achive this using assemblies. Many SNP calling pipelines will combine SNPs into a core genome alignment. My SNP pipeline Snippy will accept both assemblies and reads, but internally shreds the assemblies into fake reads rather than use the contigs natively.

One of Heng Li's past experiments was fermikit which did "rough" de novo assemblies and then aligned them to the reference, and called SNPs. One of the advantages of this is improved calling of indels.

Another experiment

Here we present ekidna which will accept either reads or contigs. Reads will be assembled in a fast and conservative method into contigs. A reference will be chosen from the contig sets, and the remainder contigs will be directly aligned to the reference using minimap2 and variants called using paftools. VCF files will then be combined into a core genome alignment suitable for building phylogenies for population analysis.

Synposis

% ekidna -t -o outdir *.fna *_R1.fastq.gz
<snip>

% figtree outdir/ekidna.nwk
<admire the SNP resolution population structure>

Installation

Conda

Install Conda or Miniconda:

conda install -c bioconda ekidna  # COMING DEC 2018

Homebrew

Install HomeBrew (Mac OS X) or LinuxBrew (Linux).

brew install brewsci/bio/ekidna  # COMING DEC 2018

Source

This will install the latest version direct from Github. You'll need to add the Ekidna bin directory to your $PATH, and also ensure all the dependencies are installed.

cd $HOME
git clone https://github.com/tseemann/ekidna.git
$HOME/ekidna/bin/ekidna --help

Interface

USAGE
  ekidna [options] -o <outdir> <SAMPLE1 SAMPLE2 SAMPLE3 ...>
SAMPLES
  Contigs    contigs.{fna,gff,gbk}[.gz] (assembled genomes)
  Reads      R1.{fq,fastq}[.gz] (only want R1)
OPTIONS
  -h         Print this help
  -v         Print version and exit
  -q         No output while running, only errors
  -k         Keep intermediate files
  -o OUTDIR  Output folder [mandatory]
  -p PREFIX  Prefix for output files [ekidna]
  -j CPUS    Number of CPU threads to use [1]
  -m MINLEN  Minimum alignment size to consider [500]
  -a ASMCMD  Assember command [skesa ...]
  -t         Also build tree

Input files

Assembled genomes or contigs

• FASTA, Genbank, EMBL, GFF ; optionally compressed with gzip, bzip2, zip

Read sequences

• FASTQ ; optionally compressed with gzip
• These will be assembled rapidly and roughly into contigs
• Only one FASTQ file is accepted ; suggest _R1 if you have paired reads

Usage

% cd test

% ls
NC_018594.fna.gz  NC_021004.fna.gz  NC_021006.fna.gz  NC_021028.fna.gz
NC_021003.fna.gz  NC_021005.fna.gz  NC_021026.fna.gz

% ekidna -o outdir *.fna.gz
<snip>

% ls outdir
ekidna.aln  ekidna.fna  ekidna.full.aln  ekidna.log  ekidna.vcf

% bcftools stats outdir/ekidna.vcf | grep ^SN
SN      0       number of samples:      6
SN      0       number of records:      34157
SN      0       number of no-ALTs:      0
SN      0       number of SNPs: 34157
SN      0       number of MNPs: 0
SN      0       number of indels:       0
SN      0       number of others:       0
SN      0       number of multiallelic sites:   275
SN      0       number of multiallelic SNP sites:       275

% ekidna -t -o outdir_with_tree *.fna.gz
<snip>

% ls outdir_with_tree
ekidna.aln  ekidna.fna  ekidna.full.aln  ekidna.log  ekidna.nwk  ekidna.vcf

% nw_indent outdir_with_tree/ekidna.nwk
(
  1:0.0053451412,
  (
    2:0.0000468773,
    3:0.0000132048
  )100:0.0047928014,
  (
    (
      (
        4:0.0000018784,
        6:0.0000041298
      )100:0.0000329960,
      7:0.0000872871
    )100:0.0018254115,
    5:0.0014513321
  )100:0.0036613244
);

Output files

File	Contents	Format
.log	log file of all the message output of the pipeline commands	ASCII text
.vcf	multisample VCF file of SNPs found	VCF
.fna	reference genome chosen from largest of input genomes	FASTA
.aln	FASTA alignment of core genome SNPs	FASTA (aligned)
.full.aln	FASTA alignment of genomes relative to the .fna reference	FASTA (aligned)
.nwk	Tree built from .full.aln using iqtree GTR+G4 model	Newick

Dependencies

• perl >= 5.18
• minimap2 + paftools.js >= 2.13
• samtools >= 1.9
• bcftools >= 1.9
• any2fasta >= 0.4
• seqtk >= 1.2
• snp-sites >= 2.0
• bedtools >= 2.0

Etymology

The name Ekidna is named for the native Australian "spiny ant-eater" called an echidna. The are coated with coarse hair and spines, and like the platypus, are egg-laying mammals (monotreme). In other words, weird but cool.

License

Ekidna is free software, released under the GPL 3.0.

Issues

Please submit suggestions and bug reports to the Issue Tracker

Author

Torsten Seemann