.__        _____.__                 
  _____|__| _____/ ____\  |   ______  _  __
 /  ___/  |/ ___\   __\|  |  /  _ \ \/ \/ /
 \___ \|  / /_/  >  |  |  |_(  <_> )     / 
/____  >__\___  /|__|  |____/\____/ \/\_/  
     \/  /_____/  

Sigflow provides useful mutational signature analysis workflows. It can auto-extract mutational signatures, fit mutation data to all/specified COSMIC reference signatures (SBS/DBS/INDEL) and run bootstrapping analysis for studying signature stability.

Any bugs or suggestions please report to GitHub issues, I will respond as soon as possible.

If you would like to use Docker, skip the following installation step and go to PART 'Use Sigflow docker image' directly.

Using Sigflow Docker image is recommended for users without experiences in programming, especially in R.

Requirements:

• R (>3.6).
• R package sigminer.
• R package docopt.

Steps:

1. Install R - follow the instructions at https://cran.r-project.org/.
2. Install R packages, run

install.packages("docopt")
install.packages("BiocManager")
BiocManager::install("sigminer", dependencies = TRUE)
# Update Sigminer version
install.packages("remotes")
remotes::install_github("ShixiangWang/sigminer")
# Install specific version by
# remotes::install_github("ShixiangWang/sigminer@v1.0.17")

3. Clone this repository, run

$ git clone https://github.com/ShixiangWang/sigflow

4. Link the R script as a executable file (command)

$ cd sigflow
$ ln -s $PWD/sigflow.R /usr/local/bin/sigflow  # You can choose another place instead of /usr/bin/sigflow

5. Try calling sigflow

sigflow -h

Maybe you need to restart your terminal.

Use specified version (recommended way):

# docker pull shixiangwang/sigflow:version, e.g.
$ docker pull shixiangwang/sigflow:1.0

NOTE: Sigflow version has no prefix v.

Current available tag versions:

Use latest version:

$ docker pull shixiangwang/sigflow:latest

The latest version uses the latest (successful build) commit from GitHub, so it may have not been prepared or fully tested. So, be careful!

Run the docker by:

$ docker run shixiangwang/sigflow

See test/test_docker.sh for examples.

If you want to go into the docker container terminal, run

$ docker run --rm --entrypoint /bin/bash -it shixiangwang/sigflow

All Sigflow commands and options are described as the following.

=================================================================
sigflow: Streamline Analysis Workflows for Mutational Signatures.

Author: Shixiang Wang (wangshx@shanghaitech.edu.cn)
Copyright: AFL@2020 [https://opensource.org/licenses/AFL-3.0]

Desc:
  There are several subcommands.
  ==
  extract - extract signatures by either automatic or semi-automatic way.
            Of note, when you use manual way, you need to run 2 times, 
            firstly you should set --manual to get signature estimation results,
            and secondly you should set --manual --number N to get N signatures.
  ==
  fit     - fit signatures in >=1 samples based on COSMIC reference signatures.
  ==
  bt      - run bootstrap signature fitting analysis in >=1 samples.
  ==
  show    - show some useful information or plots. See README for details.

Usage:
  sigflow extract --input=<file> [--output=<outdir>] [--mode=<class>] [--manual --number <sigs>] [--max <max>] [--genome=<genome>] [--nrun=<runs>] [--cores=<cores>] [--sigprofiler] [--refit] [--hyper] [--verbose]
  sigflow fit --input=<file> [--output=<outdir>] [--index=<index>] [--mode=<class>] [--genome=<genome>] [--verbose]
  sigflow bt  --input=<file> [--output=<outdir>] [--index=<index>] [--mode=<class>] [--genome=<genome>] [--nrun=<runs>] [--verbose]
  sigflow show [--isearch=<keyword>] [--index=<index> --mode=<class>] [--output=<outdir>] [--verbose]
  sigflow (-h | --help)
  sigflow --version

Options:
  -h --help     Show help message.
  --version     Show version.
  -i <file>, --input <file>       input CSV/EXCEL/MAF file or VCF directory path.
  -o <outdir>, --output <outdir>  output directory path [default: ./sigflow_result/].
  --index <index>                 reference signature index separated by comma, e.g. '1,2,3' [default: ALL].
  -m <class>, --mode <class>      extract/fit mode, can be one of SBS, DBS, ID, MAF (for three types), CN (not supported in fit subcommand) [default: SBS].
  --manual                        enable manual extraction, set -N=0 for outputing signature estimation firstly.
  -N <sigs>, --number <sigs>      extract specified number of signatures [default: 0].
  --max <max>                     maximum signature number, default is auto-configured, should >2 [default: -1].
  -g <genome>, --genome <genome>  genome build, can be hg19, hg38 or mm10, [default: hg19].
  -r <runs>, --nrun <runs>        run times of NMF (extract) or bootstrapping (bt) to get results [default: 30].
  -T <cores>, --cores <cores>     cores to run the program, large dataset will benefit from it [default: 1].
  --refit                         refit the de-novo signatures with quadratic programming or nnls (SigProfiler).
  --hyper                         enable hyper mutation handling in COSMIC signatures (not used by SigProfiler approach).
  --sigprofiler                   enable automatic extraction by SigProfiler software.
  --isearch <keyword>             search and how cancer type specific reference signature index by keyword, e.g. breast.
  -v, --verbose                   print verbose message.

=================================================================

Sigflow supports input data in VCF/MAF/CSV/EXCEL format. The file format is auto-detected by Sigflow.

For SBS/DBS/INDEL data in CSV (including TSV) or EXCEL format, the following columns typically described in MAF format are necessary:

• Hugo_Symbol: gene symbol
• Chromosome: chromosome name, e.g. "chr1"
• Start_Position: start positionof the variant (1-based)
• End_Position: end position of the variant (1-based)
• Reference_Allele: reference allele of the variant, e.g. "C"
• Tumor_Seq_Allele2: tumor sequence allele, e.g. "T"
• Variant_Classification: variant classification, e.g. "Missense_Mutation"
• Variant_Type: variant type, e.g. "SNP"
• Tumor_Sample_Barcode: sample identifier

For copy number segment data in in CSV (including TSV) or EXCEL format, the following columns are necessary:

• Chromosome: chromosome name, e.g. "chr1"
• Start.bp: start breakpoint position of segment
• End.bp: end breakpoint position of segment
• modal_cn: integer copy number value
• sample: sample identifier

Example datasets along with many example code are available in clone repository above (you can read it online at here).

The following parts give an example for each command.

Result directory of any command has the following structure.

• Files with extension.RData and .rds are R related files to reproduce the results, and can be imported into R for further analysis and visualization.
• Files with extension.pdf are common visualization results used for communication.
• Files with extension .csv are formated data tables used for inspection, communication or further analysis.

$ # Assume you have done the clone step
$ # git clone https://github.com/ShixiangWang/sigflow
$ cd sigflow/test
$ sigflow extract -i tcga_laml.maf.gz -o test_results/test_maf -m MAF -r 10 -T 4 --max 10

This will auto-extract SBS/DBS/INDEL signatures from data toga_laml.maf.gz (a gzipped MAF file from Maftools) by 10 Bayesian NMF runs with 4 computer threads and output results to directory test_results/test_maf. At default, Bayesian NMF approach is used, it starts from 10 signatures (set by --max) and reduces to a optimal signature number. If --sigprofiler is enabled, i.e.

$ sigflow extract -i tcga_laml.maf.gz -o test_results/test_maf -m MAF -r 10 -T 4 --max 10 --sigprofiler

Sigflow will use the SigProfiler to auto-extract signatures, here it will extract 2 to 10 signatures and determine the optimal solution.

NOTE, in practice, set -r to a value >=10 is recommended for auto-extraction with Bayesian NMF, >=100 for semi-automatic extraction with basic NMF and automatic extraction with SigProfiler (enabled by --sigprofiler).

Results of extract command have the following structure:

Here, no DBS records found in input data, so no corresponding result files exist.

• Tally: mutation catalogue data and plots of all samples or individual samples are stored in files/directory contains tally.

  

  

  

• Signature: signature profile (relative contribution in each signature) data and plots of all samples are stored in files contains signature.

  

  

  

• Exposure: exposure profile (relative and absolute contribution of each signature to each sample) data and plots of all samples are stored in files contains exposure.

  

  

  

• Similarity: to understand the etiologies of extracted signatures, cosine similarity analysis is applied to extracted signatures and reference signatures from COSMIC database. The result files contains similarity and best_match.

  

  

  

  

  

• Clustering: the samples can be clustered based on signature relative exposure. This analysis is done by kmeans and the results are outputed (Note, the cluster number is same as signature number).

  

  

  

$ # Assume you have done the clone step
$ # git clone https://github.com/ShixiangWang/sigflow
$ cd sigflow/test
$ sigflow fit -i tcga_laml.maf.gz -o test_results/test_fitting -m MAF

This will auto-fit input data tcga_laml.maf.gz to COSMIC SBS/DBS/INDEL signatures. Signature exposure data tables and plots are outputed.

Results of fit command have the following structure:

Here, no DBS records found in input data, so no corresponding result files exist.

legacy represents COSMIC v2 SBS signatures and SBS represents COSMIC v3 SBS signatures.

• Tally: same as results from extract command.

• Fitting: fitted relative/absolute signature exposure, reconstructed error (calculated by Frobenius norm) data and corresponding plots of all samples are stored in files contains fitting.

  

  

  Relative signature exposure in each sample.

  

  Absolute signature exposure in each sample.

  

  Visualization of relative signature exposure.

  

  Visualization of absolute signature exposure.

  

  Reconstructed error for each sample.

Bootstrapping analysis was performed to evaluate the stability of signature exposure. For a tumor, this analysis firstly resamples mutations based on the observed mutation type (component) frequency and then applies signature fitting to the bootstrapping samples. For example, if a tumor harbors 100 mutations and 20 mutations are classified into T[C>T]T, then we resample 100 mutations and the probability to assign these mutation to T[C>T]T is 0.2. If we repeat such process many times, we can estimate the confidence interval of exposure of a signature in this tumor.

More details please read paper Detecting presence of mutational signatures in cancer with confidence.

$ # Assume you have done the clone step
$ # git clone https://github.com/ShixiangWang/sigflow
$ cd sigflow/test
$ sigflow bt -i tcga_laml.maf.gz -o test_results/test_bt -m SBS -r 5

This will resample mutation catalogue of each sample based on observed mutation type frequency and run signature fitting using COSMIC SBS/DBS/INDEL signatures. The process is repeated multiple times and controlled by option -r (here is 5). This bootstrap analysis is used to estimate the instability of signature exposure. Data tables and plots of bootstrap signature exposures, errors and p values under different exposure cutoff are outputed.

NOTE, in practice, set -r to a value >=100 is recommended.

Results of bt command have the following structure:

• Tally: same as results from extract command.

• Bootstrap fitting: fitted relative/absolute signature exposure, reconstructed error (calculated by Frobenius norm of residue), signature instability data and corresponding plots of all bootstrapping samples and individual samples are stored in files/directory contains bootstrap.

  

  

  Bootstrap signature exposure distribution.

  

  For each sample, the distribution of bootstrap exposures is plotted as boxplot and the fitting result with original input data is labelled by triangle.

  

  Note, the sample data without bootstrapping process are also fitted and labelled as type = "optimal"

  

  

  The p values are calculated as the proportion of how many bootstrapping samples have exposures under specified exposure cutoff.

  

  Signature exposure instability is measured as MRSE between exposures in bootstrap samples and exposures in the original samples for each tumor/patient.

show command provides extra information to help user analyze signatures. This includes:

1. Search cancer type specific signature indices, this may help user to set the reference indices in fit and bt commands. This information could read online.
2. Generate COSMIC reference signature profiles.

For the no.1 task, one could run

$ sigflow show --isearch breast

This will generate the following output:

For the no.2 task, one could run

sigflow show --mode SBS --index 1,2,3,7a -o test_show_sig_profile

This will generate signature profile for signature 1,2,3,7. For SBS, two versions of plots exist.

COSMIC v2:

COSMIC v3:

If you use Docker to run Sigflow, you cannot directly call sigflow command. Instead, you should use sudo docker run --rm -v /your_local_path:/docker_path shixiangwang/sigflow to start a Docker container.

For example, if you want to accomplish the same task shown in extract command above, you need to run:

$ sudo docker run --rm -v /your_local_path:/docker_path shixiangwang/sigflow \
  extract -i /docker_path/tcga_laml.maf.gz \
          -o /docker_path/test_maf \
          -m MAF -r 10 -T 4 --max 10

Here,

• --rm will delete this container when this task is finished.
• -v is used for mounting your local directory /your_local_path as /docker_path in Docker image. This is important. You need to use the Docker container path in Sigflow arguments. So there must be a file called /your_local_path/tcga_laml.maf.gz exists in your computer, it will be treated as /docker_path/test_maf in the container.

• 2024-04-01: upgraded sigminer to v2.3.
• 2023-06-08: add thread controls in boostrap fit instead of using all available cores as workers.
• 2022-02-13: add troubleshooting section.
• 2021-06-17: upgraded sigminer to v2.0.2 to fix issue #31.
• 2021-04-02: upgraded sigminer to v2.0.
• 2021-03-29:
  • enhanced data check.
  • upgraded sigminer to v1.9 (the alpha version for sigminer v2).
• 2020-12-01: Another typo for hg38 reference genome build, thanks to @PalashPandey.
• 2020-11-30: Fix a typo which incorrectly assign the reference genome, thanks to @PalashPandey.
• 2020-10-19: Update citation paper.
• 2020-09-28: Copy signature estimation output pdf file from SigProfiler to result directory, release Sigflow 1.3.
• 2020-09-18: Update doc for bt command and add doc for show command.
• 2020-09-15: Release Sigflow 1.2
  • Upgrade sigminer version (v1.0.18) to fix bug when outputing results for only one signatures (#17).
• 2020-09-15: Prepare and try to release Sigflow 1.1. This is a version with its co-evolutionary R package Sigminer v1.0.17 and gold-standard de-novo signature extraction tool SigProfilerExtractor v1.0.17 as backends.
• 2020-09-14:
  • Add new command show to search cancer type specific reference signature indices and plot COSMIC signatures.
  • Support --refit in SigProfiler calling.
  • Fix a bug in identifying COSMIC v2 indices in signature fitting.
  • Upgrade sigminer version to v1.0.17.
• 2020-09-09:
  • Update README and documentation of input and usage.
• 2020-09-03:
  • Use sigminer v1.0.15 and support inputing reference signature index in fit and bt commands.
  • Allow users to decide if refit the signature exposures after de novo extraction with refit option.
  • Support matrix as input.
• 2020-08-14: Use sigminer v1.0.11 to use SigProfilerExtractor v1.0.15 to avoid issue raised from SigProfilerExtractor updates.
• 2020-08-05: Release Sigflow 1.0 and related Docker image. This version is based on Sigminer v1.0.10, R v4.0.2 and SigProfilerExtractor v.1.0.15.
  • Support SigProfiler.
  • Add verbose option.
  • Add max option.
  • Add hyper option.
  • More flexible and reasonable configuration.
• 2020-07-29: Release Sigflow 0.1 using Docker. Sigflow 0.1 is based on Sigminer v1.0.9 and R v4.0.2

There are some example data sets in this repository, you can find how to test different workflows in test/test.sh. It is time consuming to run all tests, just pick an example test similar to your task and see how it works. Before releasing a new version of Sigflow, I would run all tests to make sure they work well.

1. Error like the supplied end is > refwidth. ([#32])

The reference genome for variant calling is not (perfectly) match the specified genome in sig_tally(). If you make sure the reference genome is correct, please try finding the variant records with uncompatible position and removing them before rerun.

• For interactive analysis and visualization, please refer to its co-evolutionary R package sigminer.
• For mutation analysis, please refer to Maftools.

If you are using Sigflow fow now in academic field, please cite:

Shixiang Wang, Ziyu Tao, Tao Wu, Xue-Song Liu, Sigflow: An Automated And Comprehensive Pipeline For Cancer Genome Mutational Signature Analysis, Bioinformatics, btaa895, https://doi.org/10.1093/bioinformatics/btaa895

This software is released under Academic Free License ("AFL") v.3.0

Copyright 2020 © Shixiang Wang, Xue-Song Liu