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-# Strand-seq pipeline
+# MosaiCatcher pipeline
-> **Ongoing work**
+Structural variant calling from single-cell Strand-seq data - summarized in a [Snakemake](https://bitbucket.org/snakemake/snakemake) pipeline.
-Preliminary SV calling using Strand-seq data - summarized in a [Snakemake](https://bitbucket.org/snakemake/snakemake) pipeline.
+For Info on Strand-seq see
-Update: We just switched to a re-implementation of the SV classification. Still in the test phase
+* Falconer E *et al.*, 2012 (doi: [10.1038/nmeth.2206](https://doi.org/10.1038/nmeth.2206))
+* Sanders AD *et al.*, 2017 (doi: [10.1038/nprot.2017.029](https://doi.org/10.1038/nprot.2017.029))
+
+
+
+## Overview of this workflow
+
+This workflow uses [Snakemake](https://bitbucket.org/snakemake/snakemake) to
+execute all steps of MosaiCatcher in order. The starting point are single-cell
+BAM files from Strand-seq experiments and the final output are SV predictions in
+a tabular format as well as in a graphical representation. To get to this point,
+the workflow goes through the following steps:
+
+ 1. Read binning in fixed-width genomic windows of 50kb or 100kb via [mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher)
+ 2. Normalization of coverage in respect to a reference sample (included)
+ 3. Strand state detection ([mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher))
+ 4. Haplotype resolution via [StrandPhaseR](https://github.com/daewoooo/StrandPhaseR)
+ 5. Multi-variate segmentation of cells ([mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher))
+ 6. Bayesian classification of segmentation to find SVs using mosaiClassifier (included)
+ 7. Visualization of results using custom R plots
+
+
+## Setup
+
+While there are different options for the installation/execution (see below),
+the following steps are shared by all of them:
+
+1. **Download this pipeline.**
+
+ ```
+ git clone https://github.com/friendsofstrandseq/pipeline
+ ```
+
+2. **Configuration.** In the config file `Snake.config.json`, specify the reference genome,
+ the chromosomes you would like to analyse.
+
+3. **Add your data.** Create a subdirectory `bam/sampleName/` and place the single-cell BAM files (one file per cell) in there:
+
+ ```
+ cd pipeline
+ mkdir -p bam/NA12878
+ cp /path/to/my/cells/*.bam bam/NA12878/
+ cp /path/to/my/cells/*.bam.bai bam/NA12878
+ ```
+
+4. **BAM requirements.** Make sure that all BAM files belonging to the same sample
+ contain the same read group sample tag (`@RG SM:thisIsTheSampleName`), that they
+ are indexed and have PCR duplicates marked (the latter is especially relevant for
+ single-cell data).
+
+5. **SNP call set.** If available, specify SNV calls (VCF) in `Snake.config.json`.
+ Note that the sample name in the VCF must match the one in the BAM files.
+
+**Note:** Multiple samples can be run simultaneously. Just create different subfolders
+below `bam/`. The same settings from the `Snake.config.json` config files are
+applied to all samples.
+
+
+
+## Installation / Execution
+
+> A Snakemake version of at least 4.8.0 is required for Singularity support.
+> When only an old Snakemake version is available, remove the `singularity`
+> line in `Snakefile` and go for option 2 or 3.
+
+### Option 1: Singularity/Docker image
+
+We provide a [Docker image](https://hub.docker.com/r/smei/mosaicatcher-pipeline/)
+of this pipeline, which can be used in Snakemake together with
+[Singularity](https://singularity.lbl.gov/). This image contains all software
+(but no data) required to run MosaiCatcher.
+
+ 1. **Singularity required.** We tested this with version 2.5.1.
+
+ 2. **Run Snakemake with `--use-singularity` option.** The software inside the
+ Singularity image need to access external data, such as the reference genome.
+ These are specified in a separate config file.
+
+ We also stripped off the content of the R package
+ [BSgenome.Hsapiens.UCSC.hg38](http://www.bioconductor.org/packages/release/data/annotation/html/BSgenome.Hsapiens.UCSC.hg38.html)
+ (find it in your local R installation), which need to be made available inside
+ the image by binding these files during execution.
+ This is how the command looks like:
+
+ ```
+ # paths on the host system
+ REF="~/data/refGenomes/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna"
+ R_REF="~/R-lib/3.4.0/BSgenome.Hsapiens.UCSC.hg38/extdata/single_sequences.2bit"
+
+ snakemake \
+ --use-singularity \
+ --singularity-args \
+ "-B ${REF}:/reference.fa:ro \
+ -B ${REF}.fai:/reference.fa.fai:ro \
+ -B ${R_REF}:/usr/local/lib/R/site-library/BSgenome.Hsapiens.UCSC.hg38/extdata/single_sequences.2bit:ro" \
+ --configfile Snake.config-singularity.json
+ ```
+
+ > **Note:** Currently only hg38 is supported within the singularity inmage.
+
+ 3. **SNP call set.** External VCF files (if available) should be *copied* into a
+ subfolder of the pipeline, which can be read from within the image. Also specify
+ a relative path in `Snake.config-singularity.json`.
+
+
+
+### Option 2: Bioconda environment
-### Bioconda environment
To install the correct environment, you can use Bioconda.
- 1. **Install MiniConda:**
- In case you do not have Conda yet, it is easiest to just install [MiniConda](https://conda.io/miniconda.html).
+1. **Install MiniConda:**
+In case you do not have Conda yet, it is easiest to just install
+[MiniConda](https://conda.io/miniconda.html).
+
+2. **Create environment:**
+
+ ```
+ conda env create -n strandseqnation -f conda-environment.yml
+ source activate strandseqnation
+ ```
+
+3. **Install [mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher)**
+ and update the file paths pointing to it (and to several R scripts) in
+ `Snake.config.json`.
+
+4. **Run** `snakemake`
- 2. **Create environment:**
- ```
- conda env create -n strandseqnation -f conda-environment.yml
- source activate strandseqnation
- ```
- That's it, you are ready to go.
+### Option 3: Manual setup
-### How to use it
+1. **Install required software:**
- 1. **Install required software:**
+ * Install [mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher)
+ (*currently you will need the `develop` branch*)
+ * Install *BSgenome.Hsapiens.UCSC.hg38* from [Bioconductor](http://www.bioconductor.org/packages/release/data/annotation/html/BSgenome.Hsapiens.UCSC.hg38.html):
- * Install [mosaicatcher](https://github.com/friendsofstrandseq/mosaicatcher) (*currently you will need the `develop` branch*)
- * Get the R-scripts from [strandsequtils](https://github.com/friendsofstrandseq/strandsequtils)
- * Install BSgenome.Hsapiens.UCSC.hg38 (can be skipped of you use the Bioconda environment, see above):
```
source("https://bioconductor.org/biocLite.R")
biocLite('BSgenome.Hsapiens.UCSC.hg38')
```
- * [Strand-Phaser](https://github.com/daewoooo/StrandPhaseR) is installed automatically
- 2. **Set up the configuration of the snakemake pipeline**
+ * Install [Strand-Phaser](https://github.com/daewoooo/StrandPhaseR).
+ This is no longer installed automatically
+ * Install other required R packages
- * Open `Snake.config.json` and specify the path to the executatables
- (such as Mosaicatcher) and to the R scripts.
- * Create a subdirectory `bam/` and another subdirectory per sample (e.g.
- `bam/NA12878/`). **Multiple samples can be run together not**.
- Then copy (or soft-link) the Strand-seq single-cell libraries (one BAM
- file per cell) in there. Note that bam files need to be sorted and indexed,
- contain a read group and should have duplicates marked.
+2. **Set up the configuration of the snakemake pipeline**
- 3. **Run Snakemake**
+ * Open `Snake.config.json` and specify the path to the executatables
+ (such as Mosaicatcher) and to the R scripts.
- * run `snakemake` to compute all tasks locally
- * Alternatively, you can ask Snakemake to submit your jobs to a HPC cluster. To this end edit the `Snake.cluster.json` file according to your available HPC environment and call
+3. Run `snakemake`
- ```
- snakemake -j 100 \
- --cluster-config Snake.cluster.json \
- --cluster "???"
- ```
-
-### SNV calls
- The pipeline will run simple SNV calling using [samtools](https://github.com/samtools/samtools)
- and [bcftools](https://github.com/samtools/bcftools). If you **already have
+
+
+## Cluster support
+
+You can ask Snakemake to submit your jobs to a HPC cluster. We provided a config
+file (`cluster.json`) for this purpose, yet it might need to be adapted to your
+infrastructure.
+
+ ```
+ snakemake -j 100 \
+ --cluster-config Snake.cluster.json \
+ --cluster "sbatch --cpus-per-task {cluster.n} --time {cluster.time} --mem {cluster.mem}"
+ ```
+
+
+
+## Provide SNV calls
+
+ The pipeline will run simple SNV calling using [samtools](https://github.com/samtools/samtools) and [bcftools](https://github.com/samtools/bcftools) on Strand-seq. If you **already have
SNV calls**, you can avoid that by entering your VCF files into the pipeline.
To so, make sure the files are [tabix](https://github.com/samtools/tabix)-indexed
and specifigy them inside the `Snake.config.json` file: