Lathe

A tool for generating bacterial genomes from metagenomes with Nanopore long read sequencing

Installation

First, install miniconda3

Then install snakemake. This can be done with the following.

conda install snakemake
snakemake --version #please ensure this is >=5.4.3

Next, clone this github directory to some location where it can be stored permanently. Remember to keep it updated with git pull.

git clone https://github.com/elimoss/lathe.git

Instructions to enable cluster execution with SLURM can be found at https://github.com/bhattlab/slurm

Inputs

Alter config.yaml to provide the following:

• sample_name: Name of sample and output directory

• fast5_dirs_list: text file containing a list of absolute paths to run/fast5/* subfolders containing .fast5 files. A good way to generate this is with find -maxdepth 2 -mindepth 2 fast5_parent_dir > fodn.txt

• flowcell: flowcell code, e.g. FLO-MIN106, passed to basecaller

• kit: kit code, e.g. SQK-LSK109, passed to basecaller

• genome_size: Estimated genome size, e.g. 50m, passed to Canu.

• singularity: location (including on the internet) of a singularity image to be used for the workflow. Don't change this.

• short_reads: location of short reads to be used for Pilon polishing, or empty quotes for long-read polishing.

• use_grid: should Canu execute in distributed mode on a cluster?

• grid_options: Extra options for execution on a cluster

• canu_args: Extra options for Canu

• skip_circularization: Should circularization be omitted from the workflow?

Lathe uses the Flye assembler by default. For Canu, please specify 'canu' for the assembler parameter in the config. For cluster Canu execution, please note: if set to True, you will need to install Canu, e.g. conda install -c conda-forge -c bioconda Canu=1.8 as well as provide any additional required parameters for your job scheduler in the config.yaml file. Please see the example config file. When executing on a cluster, Canu will appear to fail, as the first process does not produce an assembly and instead spawns subsequent jobs on the cluster. Don't worry, just re-run Lathe when the assembly completes.

To execute please run the following. Please note, you must substitute a parent directory containing all of your data and working directories for /labs/.

snakemake --use-singularity --singularity-args '--bind /labs/,/scg/,/home/ ' -s /path/to/lathe/Snakefile \
--configfile path/to/modified_config.yaml --restart-times 0 --keep-going --latency-wait 30
# --profile scg #enable cluster support, highly recommended.  See above.

Outputs

The outputs generated by this workflow will look like the following:

samplename/ ├── 0.basecall │   ├── samplename.fq │   └── nanoplots ├── 1.assemble │   ├── samplename_merged.fasta │   ├── samplename_raw_assembly.fa │   ├── samplename_raw_assembly.fa.amb │   ├── samplename_raw_assembly.fa.ann │   ├── samplename_raw_assembly.fa.bwt │   ├── samplename_raw_assembly.fa.fai │   ├── samplename_raw_assembly.fa.pac │   ├── samplename_raw_assembly.fa.paf │   ├── samplename_raw_assembly.fa.sa │   ├── assemble_100m (if specified) │   └── assemble_250m (if specified) ├── 2.polish │   ├── samplename_polished.corrected.fasta │   ├── samplename_polished.fasta │   ├── samplename_polished.fasta.bam │   ├── samplename_polished.fasta.bam.bai │   ├── samplename_polished.fasta.fai │   ├── samplename_polished.fasta.misassemblies.tsv │   ├── medaka (if specified) │   ├── pilon (if specified) │   └── racon (if specified) ├── 3.circularization │   ├── 1.candidate_genomes │   ├── 2.circularization │   ├── 3.circular_sequences #circularized genomes │   ├── 4.samplename_circularized.corrected.fasta │   ├── 4.samplename_circularized.fasta │   ├── 4.samplename_circularized.fasta.bam │   ├── 4.samplename_circularized.fasta.bam.bai │   ├── 4.samplename_circularized.fasta.fai │   └── 4.samplename_circularized.fasta.misassemblies.tsv └── 5.final    ├── samplename_final.fa    └── samplename_final.fa.fai