SCAN2

Genotyper for somatic SNV and indel discovery in PTA-amplified single cells.

SCAN2 should not be used for genotyping germline mutations, as it excludes any mutation with any read support in matched bulk samples.

Installation

SCAN2 is distributed as a conda package. Installation requires the conda package management tool and a Linux-flavored OS.

Operating systems tested

• GNU/Linux, kernel version 3.10.0, CentOS 7. Note that pre-compiled SHAPEIT2 binaries are only made available for Linux systems, although in principle other phasing algorithms can be used instead.
• Ubuntu 16.04.4 LTS AWS instance.

Installing miniconda

$ wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
$ bash Miniconda3-latest-Linux-x86_64.sh
# Accept the license by typing "yes"
# Choose an install prefix (the default is often fine)
# Choose to run conda init (enter yes a second time during script)
# Log-out and back in to source .bashrc and put conda on $PATH

Installing SCAN2

Create a conda environment for SCAN2

$ conda deactivate   # The "base" environment will be active after login
$ conda create -n scan2
$ conda activate scan2

Install the SCAN2 package

$ conda install -c bioconda -c conda-forge/label/cf201901 -c jluquette scan2

Register your GATK installation

$ wget 'https://software.broadinstitute.org/gatk/download/auth?package=GATK-archive&version=3.8-1-0-gf15c1c3ef' -O GenomeAnalysisTK-3.8-1-0-gf15c1c3ef.tar.bz2
$ tar xjvf GenomeAnalysisTK-3.8-1-0-gf15c1c3ef.tar.bz2
$ gatk-register GenomeAnalysisTK-3.8-1-0-gf15c1c3ef/GenomeAnalysisTK.jar
# Test the install
$ gatk --version
# Above should print 3.8-1-0-gf15c1c3ef

Downloading external data dependencies

SCAN2 has been tested on the NCBI human reference build 37.

Download reference genome.

$ wget ftp://[email protected]/bundle/b37/human_g1k_v37_decoy.fasta.gz
$ wget ftp://[email protected]/bundle/b37/human_g1k_v37_decoy.fasta.fai.gz
$ wget ftp://[email protected]/bundle/b37/human_g1k_v37_decoy.dict.gz

Download dbSNP common variants. Note that dbSNP build 147 (common variants only) was used in the publication. However, NCBI does not guarantee long term hosting of dbSNP builds, so we recommend downloading the version of dbSNP included in the Broad's GATK resource bundle. To use other builds of dbSNP, you will need to generate a tribble index (see below).

$ wget ftp://[email protected]/bundle/b37/dbsnp_138.b37.vcf.gz
$ wget ftp://[email protected]/bundle/b37/dbsnp_138.b37.vcf.idx.gz

Download SHAPEIT's haplotype reference panel.

$ wget https://mathgen.stats.ox.ac.uk/impute/1000GP_Phase3.tgz
$ wget https://mathgen.stats.ox.ac.uk/impute/1000GP_Phase3_chrX.tgz

Unzip everything and move the chrX SHAPEIT files into the main SHAPEIT directory.

$ gunzip *.gz
$ tar xzvf 1000GP_Phase3.tgz
$ tar xzvf 1000GP_Phase3_chrX.tgz
$ mv genetic_map_chrX_* 1000GP_Phase3_chrX* 1000GP_Phase3

Running the SCAN2 demo

Download the demo chr22 BAMs.

$ wget http://compbio.med.harvard.edu/scan-snv/hunamp.chr22.bam
$ wget http://compbio.med.harvard.edu/scan-snv/hunamp.chr22.bam.bai
$ wget http://compbio.med.harvard.edu/scan-snv/il-12.chr22.bam
$ wget http://compbio.med.harvard.edu/scan-snv/il-12.chr22.bam.bai

Run SCAN2. Replace instances of /path/to/... with the paths downloaded above. This demo runs in about 5 minutes on a single core machine by restricting analysis to a 1 MB segment of chr22 and by using an impractically coarse grid for covariance function fitting.

scan2 --analysis-dir demo init
scan2 --analysis-dir demo config \
    --verbose \
    --ref /path/to/human_g1k_v37_decoy.fasta \
    --dbsnp /path/to/dbsnp_138_b37.vcf \
    --shapeit-refpanel  /path/to/1000GP_Phase3 \
    --sc-bam il-12.chr22_30M.bam \
    --bulk-bam hunamp.chr22_30M.bam \
    --regions 22:30000001-30100000
scan2 --analysis-dir demo validate
scan2 --analysis-dir demo run

See scansnv -h for more details on arguments.

After SCAN-SNV completes, single sample results are available in the Rdata file demo/scansnv/[single_cell_sample_name]/somatic_genotypes.rda. SNVs that pass SCAN-SNV's calling thresholds will have pass=TRUE in the somatic data frame (see below).

NOTE: a VCF output option is forthcoming.

# Called sSNVs can be extracted from the data frame via
R> load('demo/scansnv/[single_cell_sample_name]/somatic_genotypes.rda')
R> somatic[somatic$pass,]
# The demo should not produce any passing variants.

WARNING!

• The conda environment (named scan2 in these instructions) must always be conda activated before running SCAN2.
• Real world analyses will require parallelization.
  • On a machine with multiple cores, increasing the --joblimit parameter will run multiple parts of the analysis in parallel.
  • For clusters with distributed resource management software (e.g., SLURM), SCAN-SNV exposes Snakemake's parallelization options --cluster and --drmaa.

Using a custom dbSNP version

Generating a Tribble index for dbSNP

dbSNP VCFs must be indexed by Tribble (not tabix) for GATK. The dbSNP found in the GATK's resource bundle is already indexed. If you wish to use a different dbSNP version, the file can be indexed by igvtools.

$ conda install -c bioconda igvtools
$ igvtools index /path/to/your/dbsnp.vcf