usage: gtc_to_vcf.py [-h] [--gtc-paths GTC_PATHS [GTC_PATHS ...]]
--manifest-file MANIFEST_FILE --genome-fasta-file
GENOME_FASTA_FILE [--output-vcf-path OUTPUT_VCF_PATH]
[--skip-indels] [--log-file LOG_FILE]
[--expand-identifiers] [--unsquash-duplicates]
[--auxiliary-loci AUXILIARY_LOCI]
[--filter-loci FILTER_LOCI] [--disable-genome-cache]
[--version]
Convert GTC file to VCF format
optional arguments:
-h, --help show this help message and exit
--gtc-paths GTC_PATHS [GTC_PATHS ...]
One or more GTC files or directories to process (optional)
--manifest-file MANIFEST_FILE
Bead pool manifest for product (*.csv or *.bpm)
--genome-fasta-file GENOME_FASTA_FILE
Reference genome in fasta format
--output-vcf-path OUTPUT_VCF_PATH
Path for generation of VCF output (default is
output.vcf)
--skip-indels Skip processing of indels (default is False)
--log-file LOG_FILE File to write logging information (optional)
--expand-identifiers For VCF entries with multiple corresponding manifest
entries, list all manifest identifiers in VCF ID field
--unsquash-duplicates
Generate unique VCF records for duplicate assays
--auxiliary-loci AUXILIARY_LOCI
VCF file with auxiliary definitions of loci (optional)
--filter-loci FILTER_LOCI
File containing list of loci names to filter from
input manifest (optional)
--disable-genome-cache
Disable caching of genome reference data
--version show program's version number and exit
Input GTC files are specified with the --gtc-paths option. One or more paths may be specified with this option. If a given path corresponds to a directory, the script will identify all GTC files within that directory for processing. A combination of file and directory paths may be specified. There is a performance benefit to analyzing multiple GTC files in a single invocation of the program, as it minimizes the IO overhead of reading manifest and genome reference data to memory. When multiple input GTC files are specified, a single VCF file is produced for each input GTC file, as opposed to a single multi-sample VCF file. When no GTC file is provided, the program will still produce an output VCF file without sample genotyping information.
The --output-vcf-path option may either be a file or directory. If the argument is a directory, the program will automatically determine an appropriate name for the VCF output file created within that directory. The behavior is summarized in the following table
| # GTC files | --vcf-output-path | Behavior |
|---|---|---|
| 0 | directory | VCF filename determined from manifest filename |
| 1 | directory | VCF filename determined from input GTC file |
| 2+ | directory | VCF filenames determined from input GTC files |
| 0 | file | VCF filename determined from --vcf-output-path argument |
| 1 | file | VCF filename determined from --vcf-output-path argument |
| 2+ | file | Error |
The supplied manifest file may either be in CSV or BPM format; however, a CSV format manifest is required to generate indel records in the output VCF. When running the converter with a BPM manifest, indel processing must be explicitly disabled with the "--skip-indels" option. In either case, the manifest must provide RefStrand annotations. The GTC to VCF converter depends on the presence of accurate mapping information within the manifest, and may produce inaccurate results if the mapping information is incorrect. Mapping information should follow the implicit dbSNP standard, where
- Positions are reported with 1-based indexing
- Positions in the PAR are reported with mapping position to the X chromosome
- For an insertion relative to the reference, the position of the base immediately 5' to the insertion (on the plus strand) is given
- For a deletion relative to the reference, the position of most 5' deleted based (on the plus strand) is given
Any standard product manifest provided by Illumina will already follow these conventions.
The contig identifiers in the provided genome FASTA file must match exactly the chromosome identifiers specified in the provided manifest. For a standard human product manifest, this means that the contig headers should read ">1" rather than ">chr1". For compatibility with BaseSpace Variant Interpreter (https://www.illumina.com/informatics/research/biological-data-interpretation/variant-interpreter.html), the specified path of the reference must contain either contain the string GrCh37 or GrCh38, for build 37 and 38, respectively. Suitable whole genome FASTA files can be built with the download_reference.sh script located within the scripts directory.
In the manifest, there can be cases where the same variant is probed by multiple different assays. These assays may be the same design or alternate designs for the same locus. In the default mode of operation, these duplicates will be "squashed" into a single record in the VCF. The method used to incorporate information across multiple assays is under the latter "Output description" heading. When the "--unsquash-duplicates" option is provided, this "squashing" behavior is disabled, and each duplicate assay will be reported in a separate entry in the VCF file. This option is helpful when you are interested in investigating or validating the performance of individual assays, rather than trying to generate genotypes for specific variants. Note that if an locus has more than two alleles and is also queried with duplicated designs, the duplicates will not be unsquashed.
By default, the entire reference genome will be read into memory. Generally, this will be more efficient than reading data from the indexed reference on disk at the expense of greater memory utilization. For situations in which the genome caching is not desirable (low memory availability or a small input manifest), it is possible to disable this default behavior with the "--disable-genome-cache" option.
Certain classes of variant types (such as multi-nucleotide variants) are not currently supported in the upstream analysis software that produces GTC files. However, it is possible to query this type of variant by creating a SNP design that differentiates the specific multi-nucleotide alleles of interest. For example, if the true source sequence is
ATGC[AT/CG]GTAA
This assay could be designed as a SNP assay with the following source sequence
ATGC[A/C]NNNN
The GTC converter tool provides an option to supply a list of auxiliary records (in VCF format) to restore the true alleles for these cases in the output VCF. There are several restrictions around this function
- The auxiliary definition must be bi-allelic
- The auxiliary definition must be a multi-nucleotide variant
- There must not be multiple array assays (e.g., duplicates) for the locus
The VCF file output follows VCF4.1 format (https://samtools.github.io/hts-specs/VCFv4.1.pdf). Some additional details on output formatting:
- Genotypes are adjusted to reflect the sample ploidy. Calls are haploid for loci on Y, MT and non-PAR chromosome X for males.
- Multiple SNPs in the input manifest which are mapped to the same chromosomal coordinate (e.g. tri-allelic loci or duplicated sites) are collapsed into one VCF entry and a combined genotype generated. To produce the combined genotype, the set of all possible genotypes is enumerated based on the queried alleles. Genotypes which are not possible based on called alleles and assay design limitations (e.g. infiniumII designs cannot distinguish between A/T and C/G calls) are filtered. If only one consistent genotype remains after the filtering process, then the site is assigned this genotype Otherwise, the genotype is ambiguous (more than 1) or inconsistent (less than 1) and a no-call is returned. Please see 'test_class.py' in the 'tests' folder for unit tests and examples demonstrating the genotype merging process.
The following python packages are required. Note that the pysam package is currently not supported on the Windows platform.
| Requirement | Version |
|---|---|
| pysam | 0.9.0 |
| numpy | 1.11.2 |
| pyvcf | 0.6.8 |
An easy way to obtain a version of python with these dependencies available is to use "Miniconda". Miniconda is a minimal python installation along with a package manager ("conda") that can be used to install additional packages. First, obtain the 64-bit python 2.7 installer from https://conda.io/miniconda.html and install
bash Miniconda2-latest-Linux-x86_64.sh This will run the installer and ask you where you would like to install Miniconda. Then, install numpy, pyvcf, and pysam
conda install -c miniconda numpy=1.11.2
conda install -c bioconda pyvcf=0.6.8
conda install -c bioconda pysam=0.9.0where conda is a the package manager binary located in the installation location specified in the first step.
Copyright 2018 Illumina
Licensed under the Apache License, Version 2.0 (the "License"); you may not use this file except in compliance with the License. You may obtain a copy of the License at
http://www.apache.org/licenses/LICENSE-2.0
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