GARCOM: A user-friendly R package for genetic mutation counts

Operation

GARCOM was developed on R (≥4.0) (RRID:SCR_017299) with other dependencies and minimum versions as: data.table (≥1.12.8), vcfR (≥1.12.0), bigsnpr (≥1.4.11). Full documentation of dependencies and installation is available at GARCOM GitHub repository. There is no minimum memory (RAM) requirement, but that may vary according to the nature and size of input genetics data. GARCOM was developed on Unix platform but can also be used on other platforms (e.g. Windows, Ubuntu).

Implementation

GARCOM operates through two main functions: “gene_pos_counts” accepts PLINK¹⁰ (RRID:SCR_001757) input data, whereas “vcf_counts_SNP_genecoords” accepts VCF¹¹ input format. After reading in the data, these functions perform operations to count variants within genes/genomic regions for each individual included in the file.

output <- gene_pos_counts(recoded_genetic_data, gene_boundaries, snp_locations)

output <- vcf_counts_SNP_genecoords(recoded_genetic_data, gene_boundaries, snp_locations)

where, “output” is the object generated by GARCOM after a successful run of function; “recoded_genetic_data” is the main input file in PLINK or VCF format; “gene_boundaries”, and “snp_locations” are additional input files for gene/regions and SNP information, respectively.

Typical workflow is shown in Figure 1. In brief, the “gene_pos_counts” function will process genetic input data (“recoded_genetic_data”) generated from the PLINK software through the --recode A option. Data are read in standard matrix format using the data.table R library. For VCF files, the “vcf_counts_SNP_genecoords” function reads the VCF input file employing the extract.gt function from the vcfR library. The genotype values are read within the “GT” field.

Figure 1. Workflow for standard GARCOM functions.

In addition to the --recode A option, GARCOM requires genetic boundaries information and SNP information as shown in Table 2 and Table 3, respectively.

The output generated by GARCOM is a matrix, with M rows and N columns, where M represents the genes/genomic regions with at least one allele count and N represents the individuals. Genes/regions with zero allelic counts across all individuals are excluded from the final output. Missing values are counted as zero in final output. When no allelic counts are present in the user-defined genes, NULL value is returned. GARCOM allows missing values (NA) in input data.

In addition to the functions above described, GARCOM provides several options for user flexibility. For instance, GARCOM can be ran restricting analyses to 1) a list of genes, or 2) filter SNPs and extract individuals of interest. For instance, users can provide list of individuals using the “keep_indiv” parameter; similarly, genes can be filtered in by using the “filter_gene” parameter.

output <- gene_pos_counts(recoded_genetic_data, gene_boundaries, snp_locations, keep_indiv=mylist.txt)

output <- gene_pos_counts(recoded_genetic_data, gene_boundaries, snp_locations, filter_gene=mysetofgenes.txt)

Use cases

The input PLINK file has a matrix structure of N rows with M columns, where N rows represent individuals (one for each ID). The first six columns are family ID, individual ID, paternal ID, maternal ID, sex, and phenotype (standard output from PLINK (Table 1)). The following columns consist of the variants included in the analyses.

The input VCF file follows the standard VCF formats (please refer to the vcfR library documentation).

Toy data (gene and SNP coordinates) are shared within the package as “genecoord” and “snppos”, respectively.

Simulation

We performed simulation on real data for chromosome 1 (“CHR1”, # of variants = 23,456) and CHR22 (# of variants = 4,814) on randomly sampled individuals (N = 100, 200, 500, 1000, 5000, 10,000) extracted from whole-exome sequencing dataset as described in the study by Tosto et al.¹² Genetics data were recoded using PLINK --recode A flag. For both chromosomes we found increased memory consumption and time (Figure 2) as we increased the number of individuals included in the simulation. Memory consumption for CHR22 was significantly lower due to a smaller number of variants and genomic boundaries. Simulations were performed with 16GB memory (RAM) requested on computing cluster node.

Figure 2. Comparison of memory (in MB) and CPU time (in seconds) for CHR1 and CHR22 on different sample sizes.

Graph A represents memory consumption; graph B shows processing time in seconds for various sample sizes on CHR1 and CHR22.

All simulations were conducted on R (v4.0), data.table (v1.13.6) with default 16 threads, GARCOM (v1.40), bigsnpr (v1.6.1).

Future

VCF format can accommodate locus annotation performed by software such as ANNOVAR.¹³ To this end, GARCOM plans to accommodate annotation filters in addition to the existing ones. One challenge associated with annotated VCF is the resulting large file size; we will try to add this functionality, keeping RAM limitations and processing time in mind. We plan to add features to handle bgen format which stores large amount of genetics data, with appropriate R library (http://www.well.ox.ac.uk/~gav/resources/rbgen_v1.1.5.tgz).