Single Amplified Genome - Assembly Workflow Example (SAG-AWE)

This script is intended as a suggested workflow for the assembly and analysis of MDA sequenced 'Single Amplified Genomes' derived from Illumina Hi-/Mi-Seq paired-end libraries.

There are very few options available to the user, and most underlying programs are set with their defaults. This is intended to produce quick rough and reproducible assemblies of 100s of SAGs at a time.

SAG Assembly Workflow Example Diagram

Depending on the options you select, there are four paths to assembling your data.

• T (green) - Trimmed
• TN (blue) - Trimmed and Normalised
• TM (yellow) - Trimmed and Merged
• TNM (purple) - Trimmed, Normalised and Merged

We suggest using all three options -t, -n and -m to produce the 'best' assembly, however your library prep/design and sequencing results may work better with different options. You can run the workflow with different options in the same output directory if you wish to make comparisons.

• Red arrows indicate data requirements from previous steps
• Dotted arrows indicate reports read by MultiQC
• Grey arrows indicate future additions.

Example Usage / Help

Single Amplified Genome Assembly Workflow Example (SAG-AWE)
  Options are positional, i.e. they are run sequentially, e.g. -S must come before -q.
Input Options (required):
  -f <r1.fq|r1.fq.gz> Read Library Pair 1
  -r <r2.fq|r2.fq.gz> Read Library Pair 2
Output Options (required):
  -o <output_dir> Output Directory
Program Parameters:
  -t  Run Trim Galore!
  -n  Run Normalisation
  -m  Run Read Merging
  -s  Run Assembly
Optional Parameters:
  -C  Use to turn off Single-Cell mode in SPAdes 
  -S  Use scaffolds.fasta instead of contigs.fasta
  -L <int>  Limit contigs or scaffolds in reports to >= <int>bp
General Reports:
  -k  Run KAT Analysis (run to inform QUAST)
  -g  Run GenomeScope (requires -k)
  -p  Run Smudge Plots (requires -k)
Contig/Scaffold Specific Reports:
  -B </path/to/blast/db,/path/to/taxdump>  Run Blobtools
  -q  Run QUAST Analysis (can use -k and -B results)
  -Q  Run Qualimap Analysis (requires -B)
  -b </path/to/db1,/path/to/db2,...>  Run BUSCO with Multiple Lineages
  -M  Run MultiQC Analysis
Legacy Reports:
  -c  Run CEGMA Analysis

Example: bin/sagawe -f read1.fq.gz -r read2.fq.gz -o results -t -n -m -s -q

The -C mode allows you to turn off "single-cell" mode in SPAdes, this may be useful for some analyses. All other programs run with defaults.

Install Dependencies

The following programs will need to be installed, and be accessible from your PATH. The script has been tested on Ubuntu Linux Xenial, however it should work in other -nix environments. You will also need to make sure that the relevant environment variables for BLAST, CEGMA & AUGUSTUS are set correctly. This script does not check for dependencies and may fail without warning. Trim Galore! and SPAdes are the minimal required toolset you will need to start.

You may like to ask your local friendly bioinformatician / sys-admin to install the following programs for you in exchange for some beer or chocolate! :p Or you may try to install them yourself with conda as below.

Assembly

• Trim_Galore! (required)
  • cutadapt
  • FastQC
  • pigz
• bbmap
  • bbmerge
  • bbnorm
• SPAdes (required)

Reporting

• QUAST v4 or v5
  • glimmer
  • NCBI BLAST+
• Blobtools v1
  • BWA
  • samtools
  • NCBI BLAST+
• BUSCO v3
  • AUGUSTUS
  • bamtools
  • HMMER
• KAT
• Genomescope
• Smudgeplots
  • Jellyfish
• MultiQC

Legacy

• CEGMA
  • CEGMA is no longer supported and does not have a conda install, it is included as an option for legacy purposes only, please use BUSCO.

Databases and Variables

You will also need:

• BUSCO Lineage Datasets
• NCBI 'nt' Database
  • wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/nt.??.tar.gz
• NCBI 'taxdump' Database
  • wget ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdump.tar.gz
• NCBI 'taxdb' Database
  • wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/taxdb.tar.gz
• Environment Variables
  • export BLASTDB=/your/path/to/taxdb
  • export AUGUSTUS_CONFIG_PATH=/your/path/to/augustus/config
• GenomeScope - Manual Install Only
• SmudgePlot - Manual Install Only with Python 2

Install Dependencies with Conda

The order below seemed to work for me, however conda will give you warnings whilst solving the environment and some programs cannot be installed in these configurations, and it will take a while to install...

Python 2.7

# Broken Augustus = Broken BUSCO v2
# No smudgeplot

conda create --name sags python=2.7
conda activate sags
conda install -c bioconda multiqc jellyfish kat blast busco blobtools bwa quast spades trim-galore bbmap
export AUGUSTUS_CONFIG_PATH=/your/path/to/miniconda/envs/sags/config/
export BLASTDB=/your/path/to/taxdb

Python 3.6

# No blobtools available and multiqc from pip

conda create --name sags_p3 python=3.6
conda activate sags_p3
conda install -c bioconda busco quast jellyfish kat blast bwa spades trim-galore smudgeplot qualimap
pip install multiqc --local
export AUGUSTUS_CONFIG_PATH=/your/path/to/miniconda/envs/sags/config/
export BLASTDB=/your/path/to/taxdb
# you will need to edit the BUSCO config file with the correct paths
# /your/path/to/miniconda3/envs/sags_p3/config/config.ini

Citation

This work was initially designed during the making of [in prep]. The original scripts are available as a pre-release with the below DOI .

The script has been rewritten quite significantly since the initial release and so any further citations should also include DOI. Coming Soon