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Genetics and population analysis CNVRanger: association analysis of CNVs with gene expression and quantitative phenotypes

Summary: Copy number variation (CNV) is a major type of structural genomic variation that is increasingly studied across different species for association with diseases and production traits. Established protocols for experimental detection and computational inference of CNVs from SNP array and next-generation sequencing data are available. We present the CNVRanger R/Bioconductor package which implements a comprehensive toolbox for structured downstream analysis of CNVs. This includes functionality for summarizing individual CNV calls across a population, assessing overlap with functional genomic regions, and genome-wide association analysis with gene expression and quantitative phenotypes. Availability and implementation: http://bioconductor.org/packages/CNVRanger.


Introduction
Copy number variation (CNV) is a frequently observed deviation from the diploid state due to duplication or deletion of genomic regions . CNVs can be experimentally detected based on comparative genomic hybridization, and computationally inferred from SNP-arrays or next-generation sequencing data (Geistlinger et al., 2018). These technologies for CNV detection report, for each sample under study, genomic regions that are duplicated or deleted with respect to a reference genome. Such regions are denoted as CNV calls and are the starting point for subsequent downstream analysis. In previous work, we developed, described, and applied functionality for analyzing CNVs across a population, including association analysis with gene expression and quantitative phenotypes (da Silva et al., 2016(da Silva et al., , 2018Geistlinger et al., 2018). To allow straightforward application to similar datasets, we generalize these concepts and provide refined implementations in the CNVRanger R/Bioconductor package.

Reading and accessing CNV data
The CNVRanger package reads CNV calls from a simple file format, providing at least chromosome, start position, end position, sample ID, and integer copy number for each call (Fig. 1A). Once imported into R, the CNV data are stored for efficient representation and manipulation in Bioconductor (Huber et al., 2015) data structures as implemented in the GenomicRanges (Lawrence et al., 2013) and RaggedExperiment (Morgan and Ramos, 2017) packages.

Summarizing individual CNV calls across a population
For the analysis of CNVs in a population study, CNVRanger implements three frequently used approaches for defining recurrent regions (Fig. 1B). The CNVRuler  method trims low-density areas that would otherwise inflate the size of the resulting CNV region, by default trimming region margins that are covered by <10% of the total number of calls within a region. The reciprocal overlap (RO) procedure merges calls with sufficient mutual overlap . For example, an RO of 0.51 between calls A and B requires A to overlap at least 51% of B, and B to also overlap at least 51% of A. Particularly in cancer, it is important to distinguish driver from passenger mutations, i.e. to distinguish meaningful events from random background aberrations. The GISTIC  method identifies those regions of the genome that are aberrant more often than would be expected by chance, with greater weight given to high amplitude events (high-level copy-number gains or homozygous deletions) that are less likely to represent random aberrations.

Overlap analysis with functional genomic regions
Once recurrent CNV regions have been defined, CNVRanger allows to assess whether and to which extent these regions overlap with functional genomic regions (Fig. 1C). As a certain amount of overlap can be expected just by chance, an assessment of statistical significance is needed to decide whether the observed overlap is greater (enrichment) or less (depletion) than expected by chance. CNVRanger therefore builds on the regioneR package (Gel et al., 2015), which implements a general framework for testing overlaps of genomic regions based on permutation sampling. We use the package to sample random regions from the genome, matching size and chromosomal distribution of the CNV regions. By re-computing the overlap with the functional features in each permutation, statistical significance of the observed overlap can be assessed.

CNV-expression association analysis
The CNVRanger package implements association testing between CNV regions and RNA-seq read counts based on edgeR . For CNV regions with only one CN state deviating from the 2n reference group, this reduces to the classical 2-group comparison as previously described (Geistlinger et al., 2018). For multi-allelic CNVs (e.g. 0n, 1n, 2n), edgeR's ANOVA-like test is applied to test for expression differences in any non-diploid group with respect to the 2n group. Assuming distinct modes of action, we distinguish between (i) local effects (cis), where expression changes coincide with CNVs in the respective genes, and (ii) distal effects (trans), where CNVs supposedly affect trans-acting regulators such as transcription factors (Fig. 1D). Due to power considerations and to avoid detection of spurious effects, stringent filtering of (i) not sufficiently expressed genes, and (ii) CNV regions with insufficient sample size in groups deviating from 2n, is carried out when testing for distal effects. Local effects have a clear spatial indication and the number of genes locating in or close to a CNV region of interest is typically small; testing for differential expression between CN states is thus generally better powered for local effects and less stringent filter criteria can be applied.

CNV-phenotype association analysis
Specifically developed for CNV calls inferred from SNP-chip data, CNVRanger allows to carry out a probe-level genome-wide association study (GWAS) with quantitative phenotypes (Fig. 1E). CNV calls from other sources such as sequencing data are also supported by using the start and end position of each call as the corresponding probes. As previously described (da Silva et al., 2016), we construct CNV segments from probes representing common CN polymorphisms (allele frequency >1%), and carry out a GWAS as implemented in PLINK  using a standard linear regression of phenotype on allele dosage. For CNV segments composed of multiple probes, the segment P-value is chosen from the probe P-values, using either the probe with minimum P-value or the probe with maximum CNV frequency. This is similar to a common approach used in differential expression analysis of microarray gene expression data, where the most significant probe is chosen in case of multiple probes mapping to the same gene. Results can be displayed as for regular GWAS via a Manhattan plot.
Conflict of Interest: none declared.