MInITI: Microsatellites INstability from hIgh Throughput sequencIng

Table of Contents

• Description
• Workflows steps
• Installation
• Usage
• Performances
• Copyright

Description

This workflow classify microsatellites instability from high throughput sequencing on Illumina's instruments.

Sample classification is based on comparison with a learning model creating from a panel of stable and unstable. As consequence, it does not need a normal tissue for evaluated sample and the application come with two workflows: learn and tag.

Learn produces the learning model from a list of samples, their known classification and the list of MSI targets. It must be run on data coming from your laboratory process and the resulting model should be used to classify data generated using the same protocols.

Tag classifies loci and samples, produces confidence score for these classifications and writes an interactive report.

Workflows steps

1. MInITI learn

The learn workflow produces the learning model from a list of samples, their known classification and the list of MSI targets. It can be run only once for your panel. The model created will be one of the input of all run of the MInITI tag on the same panel with the same laboratory protocol.

Workflow (see Fig.1): * If you start from the FastQ, firsts steps are the alignment of reads and the duplicates marking. * Then, the distribution of reads lengths for each locus is retrieve. * Finally, features will be used in classifiers decision (MInITI tag) are calculated. Lengths distributions, classifiers features and status of each loci are then stored in the model file.

2. MInITI tag

The tag workflow classifies loci and samples, produces confidence score for these classifications and writes an interactive report.

Workflow (see Fig.2): * If you start from the FastQ, firsts steps are the alignment of reads and the duplicates marking. * Then, the distribution of reads lengths for each locus is retrieve. * This distribution is used by three independant classifiers to tag loci by comparison to model. Then the sample class is inferred by instability ratio on these loci. The classifiers used on loci are: * An mSINGS reimplementation, * An MSISensor-pro pro algorithm's reimplementation, * A classifier from sklearn (default: random forest) * Finally, results from all classifiers are merged and a report is produced.

Installation

1. Download code

Use one of the following:

• [user way] Downloads the latest released versions from https://github.com/bialimed/miniti/archive/releases.

• [developper way] Clones the repository from the latest unreleased version:

  git clone --recurse-submodules git@github.com:bialimed/miniti.git

2. Install dependencies

• conda (>=4.6.8):

  # Install conda wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x8664.sh && \ sh Miniconda3-latest-Linux-x8664.sh

  # Install mamba conda activate base conda install -c conda-forge mamba

More details on miniconda install here.

• snakemake (>=5.4.2):

  mamba create -c conda-forge -c bioconda -n miniti snakemake==6.15.0

More details on snakemake install here.

• In folder ${APP_DIR}/test/config set variable corresponding to genome (##${GENOME_HG38}##) in wf_learn_config.yml and wf_tag_config.yml.

• Install rules dependencies (bwa, picard, ...):

  conda activate miniti cd ${APPDIR}/test snakemake \ --cores 1 \ --use-conda \ --conda-prefix ${applicationenvdir} \ --conda-create-envs-only --snakefile ${APPDIR}/Snakefilelearn \ --configfile config/wflearn_config.yml

3. Test install

• Launch test with following command:

  conda activate miniti ${APPDIR}/test/launchwf.sh \ ${CONDAENVSDIR} \ ${WORKDIR} \ ${CLUSTERPARAMS}

Example with scheduler slurm:

  conda activate miniti
  ~/soft/miniti/test/launch_wf.sh \
    /work/$USER/conda_envs/envs \
    /work/$USER/test_miniti \
    'sbatch --partition={resources.partition} --mem={resources.mem} --cpus-per-task={threads}'

• See results in ${WORK_DIR}/tag/report/run.html.

Usage

1. MInITI learn

Important considerations

Number of Samples for model creation: To provide an accuracy compatible with medical process it is necessary to capture all the diversity on your data for each class. A large number of samples in each class can easily this goal and reduce bias. If only a small number of samples are available, at least 30 stable and 30 unstable samples per locus should be a minimum to create the model on one pathology.

Status per loci: MInITI classifies each microsatellite by comparison of these repeat lengths (from reads) to a set of stable and unstable lengths distribution on same locus. Since an unstable sample may contain both stable and unstable loci, it is best to define the status of each locus for each sample in model. However, it is possible not to differentiate the status of the loci. In this case, indicate the same status as the sample for all loci in input.known_status parameter (see configuration). Take care, this configuration is not recommended and requires two elements: * A larger number of samples in model. * A majority of samples where majority of loci with the same status as the sample.

Configuration

Copy ${APP_DIR}/config/config_learn_tpl.yml in your current directory and change values before launching. Minium required changes: * Set input.aln_pattern if you start from mak duplicates alignments or set input.R1_pattern and input.R2_pattern if you start from FastQ. * Set path to file describing status in input.known_status. This TSV file contain status (MSI or MSS or Undetermined) of each analysed locus (columns) for each sample (rows). See example test/config/known_status.tsv. * Set path to the file containing locations of targeted microsatellites in reference.microsatelites (format BED). * Set path to the reference genome file in reference.sequences (format Fasta). It must be indexed (FAI) and if start from FastQ it must be indexed for BWA.

Details on each parameter can be found in configlearntpl.yml.

launch command

conda activate miniti
snakemake \
  --use-conda \
  --conda-prefix ${application_env_dir} \
  --jobs ${nb_jobs} \
  --jobname "miniti.{rule}.{jobid}" \
  --latency-wait 100 \
  --snakefile ${application_dir}/Snakefile_learn \
  --cluster "sbatch --partition={resources.partition} --mem={resources.mem} --cpus-per-task={threads}" \
  --configfile workflow_parameters.yml \
  --directory ${out_dir} \
  > ${out_dir}/wf_log.txt \
  2> ${out_dir}/wf_stderr.txt

Output directory

The main elements of the output directory are the following:

out_dir/
 ...
 microsat/
     microsatModel_info.tsv
     microsatModel.json

${out_dir}/microsat/microsatModel_info.tsv contains number of sample kept in model for each locus and status (see Fig.3).

${out_dir}/microsat/microsatModel.json contains lengths distributions, pre-calculated classifiers features and associated status in computer readable format defined by AnaCore library.

2. MInITI tag

Configuration

Copy ${APP_DIR}/config/config_tag_tpl.yml in your current directory and change values before launching. Minium required changes: * Set path to learning model generated by MInITI learn in classifier.model. * Set the instability threshold for your panel in classifier.sample.instability_threshold. * Set input.aln_pattern if you start from mak duplicates alignments or set input.R1_pattern and input.R2_pattern if you start from FastQ. * Set path to the file containing locations of targeted microsatellites in reference.microsatelites (format BED). * Set path to the reference genome file in reference.sequences (format Fasta). It must be indexed (FAI) and if start from FastQ it must be indexed for BWA.

Details on each parameter can be found in configtagtpl.yml.

launch command

conda activate miniti
snakemake \
  --use-conda \
  --conda-prefix ${application_env_dir} \
  --jobs ${nb_jobs} \
  --jobname "miniti.{rule}.{jobid}" \
  --latency-wait 100 \
  --snakefile ${application_dir}/Snakefile_tag \
  --cluster "sbatch --partition={resources.partition} --mem={resources.mem} --cpus-per-task={threads}" \
  --configfile workflow_parameters.yml \
  --directory ${out_dir} \
  > ${out_dir}/wf_log.txt \
  2> ${out_dir}/wf_stderr.txt

Output directory

The main elements of the output directory are the following:

out_dir/
 ...
 report/
     data/
    |    sample-A_stabilityStatus.json
     ...
     run.html
     sample-A.html

${out_dir}/report/data/${sample}_stabilityStatus.json contains classification information about sample in computer readable format defined by AnaCore library.

${out_dir}/report/${sample}.html (see Fig.4) is an interactive report to inspect: * Sample classification and confidence score from all classifiers. * Loci sequencing depths, distribution lengths profile (see Fig.5), classifications and confidence score from all classifiers.

Performances

Performance was evaluated on a dataset from 120 colorectal cancer patients. Samples were sequenced with a targeted panel (mutation hotspots and MSI) from FFPE block. The results summarized in assessment/report.html. Commands and configurations used in evaluation process can be found in assessment folder.

Copyright

2022 Laboratoire d'Anatomo-Cytopathologie du CHU Toulouse