Snakemake Wrapper: Trycycler Genome Assembly

This is a snakemake wrapper to run trycylcer. The sub-workflows are divided into three different steps, following the original author's instruction: https://github.com/rrwick/Trycycler/wiki. See step 4 for how to run the subworkflows.

Tutorial

Step 1: Clone the workflow

Clone this repository to your local system, into the place where you want to perform the data analysis.

git clone [email protected]:matinnuhamunada/trycycler_snakemake_wrapper.git
cd trycycler_snakemake_wrapper

Step 2: Get example data

mkdir -p data/raw/GCF_000012125
wget -O data/raw/GCF_000012125/23754659.tar.gz https://bridges.monash.edu/ndownloader/files/23754659
(cd data/raw/GCF_000012125 && tar -xvzf 23754659.tar.gz)

Step 3: Configure workflow

Setting Up Your Samples Information

Configure the workflow according to your needs via editing the files in the config/ folder. Adjust config.yaml to configure the workflow execution, and samples.tsv to specify the strains to assemble. The file units.tsv contains the location of the paired illumina and nanopore reads for each strain.

samples.tsv example:

strain	description
GCF_000012125	Example

units.tsv example:

strain	unit	illumina_reads	nanopore_reads
GCF_000012125	1		data/raw/GCF_000012125.1

Further formatting rules will be defined in the workflow/schemas/ folder.

Step 3: Install Snakemake

Installing Snakemake using Mamba is advised. In case you don’t use Mambaforge you can always install Mamba into any other Conda-based Python distribution with:

conda install -n base -c conda-forge mamba

Then install Snakemake with:

mamba create -c conda-forge -c bioconda -c panoptes-organization -n snakemake snakemake panoptes-ui

For installation details, see the instructions in the Snakemake documentation.

Step 4: Executing the workflow

Activate the conda environment:

conda activate snakemake

Run panoptes to monitor jobs:

tmux new-session -A -s panoptes \; send -t panoptes "conda activate snakemake && panoptes" ENTER \; detach -s panoptes # run panoptes in background at http://127.0.0.1:5000

Do a dry-run:

snakemake --snakefile workflow/Snakefile-assembly --use-conda --cores 8 --wms-monitor http://127.0.0.1:5000 -n

We can then open http://127.0.0.1:5000 to monitor our jobs

See the Snakemake documentation for further snakemake CLI details.

Results structure

The results can be found in the data folder and are separated into three stages: raw, interim, and processed. Further, the results will be splitted into the three steps.

Part 1 - Generating Assemblies

Running snakefile

This step generates multiple assemblies as described in: https://github.com/rrwick/Trycycler/wiki/Generating-assemblies

snakemake --snakefile workflow/Snakefile-assembly --use-conda --cores <n_cores> --wms-monitor http://127.0.0.1:5000 

Evaluating Assemblies

The first step in Trycycler is to generate assemblies from the subsampled data. Here, the subsets were assembled with three different assemblers. We can see that all assemblers agrees to generate a circular chromosome and a plasmid. | data/processed/GCF_000012125/01_trycycler_assembly/GCF_000012125_graphs.png |

Part 2 - Clustering Contigs

Running the snakefile

This step clusters the assemblies into per-replicon groups as described in: https://github.com/rrwick/Trycycler/wiki/Clustering-contigs

snakemake --snakefile workflow/Snakefile-cluster --use-conda --cores <n_cores> --wms-monitor http://127.0.0.1:5000 

This step also generate data/interim/02_trycycler_cluster/cluster.yaml which should be copied to the config folder in order to proceed to the next step.

cp data/interim/02_trycycler_cluster/cluster.yaml config/cluster.yaml

NOTE: You can select or drops the bad contigs or clusters that will be run in the next step. See below about evaluating the clusters.

Evaluating Contig Clusters

The second step in Trycycler is to cluster the contigs. Here we can see how the chromosome and the plasmid are grouped in different cluster. Each cluster should have similar length and read depths. | data/processed/GCF_000012125/02_trycycler_cluster/GCF_000012125_cluster.png |

As we can see in the figure, the plasmid sizes after the assembly is a bit weird. There are some contigs with 7500 bp and some with 15kb and 22kb. Open up the data/interim/02_trycycler_cluster/cluster.yaml and remove the contigs from cluster 2 which have length > than 8 kb.

GCF_000012125:
  cluster_001:
    - A_contig_1
    - B_utg000001c
    - C_Utg422
    - D_contig_1
    - E_utg000001c
    - F_Utg418
    - G_contig_1
    - H_utg000001c
    - I_Utg428
    - J_contig_1
    - K_utg000002c
    - L_Utg448
  cluster_002:
    - B_utg000002c
    - E_utg000002c
    - H_utg000002c
    - I_Utg430
    - K_utg000001c
    - L_Utg450

Part 3 - Generating Consensus

This step summarizes step 3, 4, 5, and 6 in the Trycycler wiki and generate the consensus contig sequence as described in: https://github.com/rrwick/Trycycler/wiki/Generating-a-consensus

snakemake --snakefile workflow/Snakefile-consensus --use-conda --cores <n_cores> --wms-monitor http://127.0.0.1:5000 

The final assembly can be found in data/processed/GCF_000012125/03_trycycler_consensus/GCF_000012125.fna

Step 5: Investigate results

TO DO