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The functions provided by this package can be categorized into five sections: Conversion; Summary; Annotation; Comparison; and Visualization. The most useful features provided are: integrating summarized results, generating lists of CNVRs, annotating the results with known gene positions, plotting CNVR distribution maps, and producing customized visualizations of CNVs and ROHs with gene and other related information on one plot (Figure 1). This package supports a range of customizations, including the color, size of high-resolution figures, and choice of output folder to avoid conflict between the results of different runs. Where applicable, output files are compatible with other software such as PennCNV (Wang et al., 2007), Plink (Chang et al., 2015), or DAVID annotation tools (Jiao et al., 2012).

The conversion section handles the conversions of genomic positions between two reference genomes, and provides two functions. convert_map is designed to compare SNP map files for two different reference genomes, matching by SNP name, and produce SNP maps in a format suitable for use by convert_coord. The function also reports the density of SNPs by chromosome. convert_coord is designed to convert the physical positions of genomic intervals based on a given SNP map file. Currently, the function is limited to inputs generated by convert_map, and can only convert the coordinates for intervals on the same type of SNP chip. Converting coordinates may change the total length of the intervals, as the positions and orders of the SNPs on the chromosome will potentially differ between various reference genomes; therefore, the function produces a table that summarizes how many intervals were converted successfully, and reports on the differences in length between the converted and original intervals.

The summary section contains a group of functions to summarize CNV results, generate CNVRs, and make CNVR distribution maps from CNV results. There is also a collection of functions to summarize ROH results, report frequencies of ROH regions, inbreeding coefficient by different length groups and to generate haplotypes on interesting ROH regions.

The functions used for reporting CNV results include clean_cnv, summary_cnv_plot, and call_cnvr. clean_cnv takes a CNV list from PennCNV and CNVPartition and reformats it into a standard format for use in the functions listed below. cnv_summary_plot generates a range of summary plots, aggregating CNV results by length group, CNV type, chromosome, and individual. call_cnvr generates CNV regions as the union of sets of CNVs that overlap by at least one base pair (Redon et al., 2006). This function will output three tables: (a) the list of CNVRs, containing the number of CNVs and number of samples in each CNVR that can reflect the frequency of CNVRs; (b) a brief summary table showing numbers of CNVRs by length and type (Deletion, Duplication, and Mixed, where Mixed indicates that both duplications and deletions are found within the CNVR); and (c) the total length and number of CNVRs on each chromosome.

roh_window will report: a table of high frequency ROH regions on the autosomes that passed the common frequency threshold, a table containing inbreeding coefficients by different length groups of each individual, a brief summary of the total numbers and lengths of ROHs in length groups, and a plot of high frequency ROH regions by chromosome. The inbreeding coefficients are calculated as F_roh = (∑L_roh)/(∑L_auto) (McQuillan et al., 2008), where ∑L_roh is the total length of ROH, and ∑L_auto is the total length of autosomes. Other functions in this group include prep_phased, closer_snp, and get_haplotype; see the package vignette for more information (Jinghang et al., 2021).

The annotation section facilitates downloading and formatting reference gene lists, and annotating genes on genomic intervals. get_refgene will automatically download a reference gene list and invoke clean_ucsc and clean_ensgene from UCSC (Navarro Gonzalez et al., 2021) websites for human, cow, sheep, pig, horse, chicken or dog species, then remove the duplicated genes and report the standard format as output. call_gene is used to report how many genes are located in the given genomic intervals. The frequency of genes is calculated from the number of samples that has the same gene annotated in its CNVs.

The comparison section consists of functions for comparing sets of CNVs (compare_cnv), CNVRs (compare_cnvr), gene frequency lists (compare_gene), and other intervals (compare_interval). These functions were implemented using the foverlaps function in the data.table R package (Dowle et al., 2019). compare_gene can produce consensus gene lists, given lists of genes present in CNVRs in multiple studies. The remaining functions report numbers, lengths, and proportions of overlapping intervals (CNVs, CNVRs, etc.) on a population and individual basis.

Finally, twelve functions in HandyCNV are included in the visualization section; of these, five produce plots as a subset of their output, and have been mentioned previously: cnv_summary_plot, roh_window, compare_cnv, compare_cnvr, and convert_map. The remaining visualization functions mainly focus on customizing and integrating the plotting of all information related to CNV, ROH, and high frequency CNVR: these are cnvr_plot, plot_gene, cnv_visual, roh_visual, plot_cnvr_panorama, plot_snp_density, and plot_cnvr_source. These functions are described in the package vignette (Jinghang et al., 2021).

The CNV result in this example was cited from a study published in 2020 which comprised 268 microarrays samples in a human population in Brazil (de Godoy et al., 2020). In this example, we will introduce how to prepare the standard CNV list, then produce brief summary, generate CNVRs, annotate genes and visualize CNVs. Figure 4 presents the code used in example 1, the R script can be found in Supplementary File 1.

To replicate this example, we first need to download the dataset “Table S1 – Detailed information about all CNVs analyzed in our sample” (de Godoy et al., 2020) and save the sheet “All array platforms’ CNVs” as.csv format file. Then use read.csv to load the CNV list and select the columns required by cnv_clean (see Figure 5C).

A formatted clean CNV list will return as an object named “clean_cnv” in working environment, and a brief summary table of CNV (see Figure 5D) will be written out after executing cnv_clean.

We then take a quick look at the CNV distribution by reading the “clean_cnv” list as input and customizing parameters in cnv_visual. In example, we first set “chr_id = 14” to visualize CNVs distribution on chromosome 14 (see Figure 5E), then zoom into the region with higher frequency CNVs (see Figure 5I) by setting “start_position = 105” and “end_position = 110.” Visualizing other chromosomes or regions and changing the colors of copy numbers can easily be done by adjusting the relevant arguments.

The CNV summary plot (see Figure 5A) can be plotted via cnv_summary_plot by taking “clean_cnv” as input. The CNVR list (see Figure 5F) is generated using call_cnvr by taking the “clean_cnv” file as input, producing a brief summary table of CNVR (see Figure 5G) that will be saved in the working directory in the meantime. The CNVR distribution map (see Figure 5B) is generated via cnvr_plot by loading the CNVR list.

For gene annotation steps, the reference gene list can be downloaded and formatted by assigning the genome version argument in get_refgene. Then the genes annotation list of CNV or CNVR are generated by running call_gene. Three input files need be assigned in the function: the clean CNV file (“clean_cnv”), the CNVR list (“cnvr”), and the reference gene list (“human_hg19”); the gene frequency list (see Figure 5J) will be returned as an object in the R environment. We can plot all the high frequency CNVRs with gene annotation results (see one example plot in Figure 5H) at the same time through cnvr_plot by reading “cnvr,” “clean_cnv” and reference gene list (“human_hg19”) and setting the “sample_size” and “common_cnv_threshold” arguments.

Finally, we can extract Sample IDs of CNVs that contain genes of interest (see Figure 5K) using get_samples, by loading the CNV annotation list generated by call_gene and assigning the gene name to the “gene_name” argument.

Since this example only contains one CNV result in one reference genome, the functions in the comparison and conversion sections are not applicable in this example. Users of these functions can browse the vignette of this package from the Github repository (Jinghang et al., 2021).

The genotype data used to detect ROH in this example is from the work of Velie et al. (2016) and contains 285 horse samples. This example aims to present how to use the functions in HandyCNV to analyze ROHs. This example includes ROH detection by Plink 1.9 (Chang et al., 2015) and genotype phasing by Beagle 5.1 (Browning et al., 2018). Figure 6 presents the code used in example 2; the R script can be found in Supplementary File 2.

To run this example, we first need to prepare the genotype data. The genotype files are read using the fread function (Dowle et al., 2019). Because the original ped file does not match the format required by Plink 1.9, we insert a sequential column of family IDs, plus placeholder columns of zeroes for the father, mother, and sex code by using data.frame and cbind functions (R Core Team, 2020). Before testing the ROH, the map file was loaded as the input file in plot_snp_density to get a brief summary and visualization of SNP density (Figure 7A). The jpeg and dev.off functions (R Core Team, 2020) are used to save the plot.

Then, we invoke Plink 1.9 (Chang et al., 2015) by shell (R Core Team, 2020) from R Studio (Team, 2021) to generate binary genotype files and call ROH. For Windows operating systems, ensure that the plink.exe file is either in the current directory or accessible via the PATH system variable. To run Plink 1.9 on other operation system, please refer to the Plink website (Chang et al., 2015).

Once we get ROH results, we can run roh_window, which takes a “plink.hom” file as input to report the brief summary of ROH by length group (see Figure 7B), high frequency ROH regions (see Figure 7D), ROH frequency distribution plot (see Figure 7G), and to calculate the ROH based inbreeding coefficient (Figure 7K).

In this example, we present visualizations of ROH on the whole of chromosome 22 (see Figure 7C) and on the 22.81–23.22 Mb region on chromosome 22 (see Figure 7E) via roh_visual, which needs to load the “plink.hom” data set as input. The “chr_id” or “target_region” arguments are available to customize visualization, alongside additional arguments to customize the colors of ROHs.

The horse reference gene list (“quaCab2”) was downloaded from the UCSC website (Navarro Gonzalez et al., 2021) by get_refgene. The genes located in the high frequency ROH regions (see Figure 7F) were annotated via call_gene, which requires loading the reference gene list (“quaCab2”) and the high frequency ROH regions file that was generated by roh_window. Since we have the reference gene list, we can visualize ROH region with genes (see Figure 7H) via roh_visual by assigning the clean ROH file (“clean_roh = clean_roh”), target ROH region [“target_region = c (1, 139.6, 141.6)”] and reference gene lists (“refgene = equaCab2”). We can also visualize ROHs in terms of the gene we are interested in: here, we are looking at the GABPB1 gene, first, exacting the physical position of this gene from the reference gene list (“equaCab2”) using the “filter” and “select” functions (Wickham et al., 2019), then using visual_roh to load the ROH file (“plink.hom”) as input and assigning the gene position to the “target_region” argument to present the plot (see Figure 7E). We can write a loop (R Core Team, 2020) of visual_roh to plot all regions with genes annotated by iterating over the high frequency ROHs that contain genes.

To get the haplotype of the genes need the phased genotype files. Here, we take chromosome 1 as example to present how to use Plink 1.9 (Chang et al., 2015) and Beagle 5.1 (Browning et al., 2018) to phase the genotypes. The shell (R Core Team, 2020) function is used to invoke plink (Chang et al., 2015) to generate the VCF format genotype file, then to invoke beagle (Browning et al., 2018) to phase the genotypes from Rstudio (Team, 2021). For Windows operating systems, ensure that the plink and java executables are either in the current directory or accessible via the PATH system variable. Likewise, adjust the path to the Beagle JAR file as required for your operating system. For instructions on installing and running Beagle 5.1, refer to their manual (Browning et al., 2018).

Finally, we take GABPB1 as an example to show how to get the haplotypes. First, we use prep_phased to load the phased genotype file (phased_geno = “orse_chr1_phased.vcf.gz”) that was generated by Beagle, and set the “convert_letter” argument as “TRUE” to convert the genotype file into the standard format used by HandyCNV (returned as “geno_chr1”). Second, we use closer_snp to extract the gene’s position (returned as “GABPB1_pos”) from the SNP map file, which requires the SNP map file (provided using the “phased_input” argument), and to assign the gene’s physical position we got from reference gene list to the “chr,” “start,” and “end” arguments, respectively. Finally, we use get_haplotype to get the haplotype information (see Figures 7I,J) for the GABPB1 gene by assigning the formatted phased genotype list (“geno_chr1”) to the “geno” argument and assigning the gene’s position (“GABPB1_pos”) to the “pos” argument.