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QuasiModo: Assessing viral genomic analysis methods on HCMV strain mixture

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QuasiModo - Quasispecies Metric Determination on Omics

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Strain-level assembly and variant calling benchmarking based on sequencing data of mixed strain samples for HCMV

This repository contains the scripts and pipeline that reproduces the results of the HCMV benchmarking study. In this study we evaluated genome assemblers and variant callers on 10 in vitro generated, mixed strain HCMV sequence samples, each consisting of two lab strains in different abundance ratios. This tool can also be used to evaluate assemblies and variant calling results on other similar datasets.

In this benchmarking study: variants callers BCFtools (v1.9), VarScan (v2.4.3), Freebayes (v1.2.0), LoFreq (v2.1.3.1), CLC Genomics Workbench (v11.0.1) were evaluated. For the assembly benchmarking, ABySS (v2.1.4), megahit (v1.1.3) , IDBA (v1.1.3), SPAdes (v3.12.0), Ray (v2.3.1), Tadpole (v37.99) were assessed. The haplotype reconstruction program Savage (v0.4.0) was also evaluated.

Prerequirements

To reproduce the output, you need to use Bioconda.

Please follow the instruction here to install Bioconda. And then you need to install snakemake, csvtk and Python package click:

conda install snakemake=5.3.0
conda install csvtk=0.18.2
conda install click=7.0

After this has been done, download the pipeline onto your system:

git clone https://github.com/hzi-bifo/Quasimodo.git

Download the dataset and prepare the sample list file (optional, only in the case you specify --slow option)

If you want to run the whole analyses on the reads you should download the reads. All sequencing data can be obtained from ENV with accession number: PRJEB32127. Download the sequencing data into a directory, for example: <your project path>/data/seqs/reads:

# Create the dir for the reads
mkdir -p <your project path>/data/seqs/reads
# Download the sequencing data
cd HCMV_benchmark
wget -i data/PRJEB32127.txt -P <your project path>/data/seqs/reads
  • Provide the sample list config/sample_list.tsv. The list is a tab delimited text file, and echo row is one sample. The format is as follows:
sample	r1	r2
TA-0-1	../HCMV_benchmark_output/data/seqs/reads/TA-0-1.qc.r1.fq.gz	../HCMV_benchmark_output/data/seqs/reads/TA-0-1.qc.r2.fq.gz
TA-1-0	../HCMV_benchmark_output/data/seqs/reads/TA-1-0.qc.r1.fq.gz	../HCMV_benchmark_output/data/seqs/reads/TA-1-0.qc.r2.fq.gz
TA-1-10	../HCMV_benchmark_output/data/seqs/reads/TA-1-10.qc.r1.fq.gz	../HCMV_benchmark_output/data/seqs/reads/TA-1-10.qc.r2.fq.gz
...

Please modify the paths to the sequencing files which you have downloaded accordingly. In this example, the <your project path> is ../HCMV_benchmark_output and the reads are in the ../HCMV_benchmark_output/data/seqs/reads.

! Due to the high computational and time cost, by default this program do not run the whole benchmark for HCMV dataset from scratch (based on reads), instead it benchmarks the variant call and assembly based on the VCF files and scaffolds provided within this program under data directory.

Adapt the configuration file

All the paths must be either relative path to the parent directory of config folder or absolute path.

The genome sequences of HCMV strains and Phix are included in this repo in the ref directory.

  • Modify the config/config.yaml:
samplesDesc: config/sample_list.tsv # leave it empty if you do not use --slow option
MerlinRef: ref/Merlin.BAC.fa # path to Merlin genome
TB40ERef: ref/TB40E.GFP.fa # path to TB40 genome
AD169Ref: ref/AD169.BAC.fa # path to AD169 genome
PhixRef: ref/Phix.fa # path to Phix genome
outpath: <your output path> # the directory for outputs
threads: 2 # number of cores to use
runOnReads: false # Run the whole analyses on reads. Controlled by the `--slow` option

Run the benchmarking

All evaluation can be launched with run_benchmark.py

The parameters for run_benchmark.py:

Usage: run_benchmark.py [OPTIONS] COMMAND [ARGS]...

Options:
  --version      Print the version.
  --help         Show this message and exit.

Commands:
  hcmv     Benchmarking for HCMV dataset
  vareval  Variant calling benchmark for customized dataset
  asmeval  Assembly benchmark for customized dataset

This program consists of three subcommands: hcmv, vareval, asmeval. The first one is used for the benchmarking on our HCMV datasets. And the other two are for the variant call and assembly evaluation on customized datasets.

The argumentrs and options in the hcmv command:

Usage: run_benchmark.py hcmv [OPTIONS]

  Benchmarking for HCMV dataset

Options:
  -o, --outpath PATH              The directory where to put the results and
                                  figures. The path can be specified either in
                                  the CLI as argument or in the config file.
  -c, --conda_prefix PATH         The prefix of conda ENV. [default: in the
                                  working directory].
  -t, --threads INTEGER           The number of threads to use.  [default: 2]
  -d, --dryrun                    Print the details without run the pipeline.
                                  [default: False]
  -e, --evaluation [all|variantcall|assembly]
                                  The evaluation to run.  [required]
  -s, --slow                      Run the evaluation based on reads, which is
                                  very slow. By default, the evaluation will
                                  be based on the VCF and contig files
                                  provided within this software. If this
                                  parameter is on, this software will run all
                                  the analyses to generate outputs based on
                                  reads for benchmarking which is very time
                                  consuming.  [default: False]
  --help                          Show this message and exit.

Evaluate the assembly and haplotype reconstruction

python3 run_benchmark.py hcmv -e assembly -t 10 -c ~/miniconda3/envs

Please use -c parameter to specify the desired path to create the conda ENVs. The conda ENVs will be created under the path of the program by default. The program may take 1 hour to create the ENV used in this benchmarking for the first time.

If you expect to the benchmarking based on the reads, you need to specify the --slow or -s option which allows you to generate the assembly results from reads.

Assess variant callers and analyze the mutation context of identified variants

python3 run_benchmark.py hcmv -e variantcall -t 10 -c ~/miniconda3/envs

If you wish to the benchmarking based on the reads, you need to specify the --slow or -s option which allows you to generate the variant calling results from reads.

All in one:

python run_benchmarking.py hcmv -e all -t 10 -c ~/miniconda3/envs

Produce the rardar plot for assembly evaluation

Since R package ggradar for making radar plot is not available in conda, CRAN and Bioconductor, so the script has to be run manually.

Rscript --vanilla scripts/assembly_radarplot.R -f <the assembly_metaquast_scaled.tsv file produced by the pipeline QuasiMode> [-o the output file name for the radar plot]
Options:
	-f CHARACTER, --file=CHARACTER (required)
		assembly_metaquast_scaled.tsv file generated by QuasiModo

	-o CHARACTER, --out=CHARACTER
		output file name of the radar-plot [default= assembly_metaquast_radarplot.pdf]

	-h, --help
		Show this help message and exit

Note: Please install devtools, dplyr, tidyr ,ggplot2, optparse and ggradar before using the script.

The output structure

└── results
    ├── asssembly
    │   ├── abyss
    │   ├── idba
    │   ├── megahit
    │   ├── metaspades
    │   ├── ray
    │   ├── savage
    │   ├── spades
    │   └── tadpole
    ├── final_figures
    ├── final_tables
    ├── metaquast
    │   ├── summary_for_figure
    │   ├── TA
    │   └── TM
    └── snp
        ├── callers
        └── nucmer

For full run based on reads (-s option on)

├── data
│   └── seqs
│       ├── bam
│       ├── cl_bam
│       ├── cl_fq
│       ├── pear_merge
│       ├── pileup
│       └── reads
├── reports
│   ├── benchmarks
│   ├── bwa
│   └── picard
└── results
    ├── asssembly
    │   ├── abyss
    │   ├── idba
    │   ├── megahit
    │   ├── metaspades
    │   ├── ray
    │   ├── savage
    │   ├── spades
    │   └── tadpole
    ├── final_figures
    │   └── refined
    ├── final_tables
    ├── metaquast
    │   ├── summary_for_figure
    │   ├── TA
    │   └── TM
    └── snp
        ├── callers
        └── nucmer

Run the benchmark on your own datasets

Evaluate the assembly or haplotype reconstruction

You can specify the parameters and files in config/customize_data.yaml:

vcfs: ## comma-separated a flist of VCF files to evaluate
scaffolds: ## comma-separated a list of contig/scaffold fasta files to evaluate
refs: ## comma-separated list of reference genomes
outpath: ../custom_benchmark_out/
threads: 2

The arguments and options of asmeval command:

Usage: run_benchmark.py asmeval [OPTIONS]

  Assembly benchmark for customized dataset

Options:
  -o, --outpath PATH       The directory where to put the results and figures.
                           The path can be specified either in the CLI as
                           argument or in the config file.
  -c, --conda_prefix PATH  The prefix of conda ENV. [default: in the working
                           directory].
  -t, --threads INTEGER    The number of threads to use.  [default: 2]
  -d, --dryrun             Print the details without run the pipeline.
                           [default: False]
  -s, --scaffolds TEXT     Comma-separated list of scaffold files. Please
                           quote the whole parameter if there is any white
                           space the file names. The files can be specified
                           either in the CLI as argument or in the config
                           file.
  -r, --refs TEXT          Comma-separated list of reference genome files.
                           Please quote the whole parameter if there is any
                           white space the file names. The files can be
                           specified either in the CLI as argument or in the
                           config file.
  --help                   Show this message and exit.
  • Run the benchmarking
python3 run_benchmark.py asmeval -t 10 -c ~/miniconda3/envs \
        -s "<comma-separated list of scaffolds>" \
        -r "<comma-separated list of reference genomes>" \
        -o <output directory>

Assess variant callers

The arguments and options of vareval command:

Usage: run_benchmark.py vareval [OPTIONS]

  Variant calling benchmark for customized dataset

Options:
  -o, --outpath PATH       The directory where to put the results and figures.
                           The path can be specified either in the CLI as
                           argument or in the config file.
  -c, --conda_prefix PATH  The prefix of conda ENV. [default: in the working
                           directory].
  -t, --threads INTEGER    The number of threads to use.  [default: 2]
  -d, --dryrun             Print the details without run the pipeline.
                           [default: False]
  -v, --vcfs TEXT          Comma-separated list of VCF files. Please quote the
                           whole parameter if there is any white space the
                           file names. The files can be specified either in
                           the CLI as argument or in the config file.
  -r, --refs TEXT          Comma-separated list of reference genome files.
                           Please quote the whole parameter if there is any
                           white space the file names. The files can be
                           specified either in the CLI as argument or in the
                           config file.
  --help                   Show this message and exit.
  • Run the benchmarking
python3 run_benchmark.py vareval -t 10 -c ~/miniconda3/envs \
        -v "<comma-separated list of VCF files>" \
        -r "<comma-separated list of reference genomes>" \
        -o <output directory>

Citation

Deng ZL, Dhingra A, Fritz A, Götting J, Münch PC, Steinbrück L, Schulz T, Ganzenmueller T, McHardy AC. Evaluating assembly and variant calling software for strain-resolved analysis of large DNA-viruses. Briefings in Bioinformatics. 2020:7. https://doi.org/10.1093/bib/bbaa123

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