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Affordable genome sequencing technologies promise to revolutionize the field of human genetics by enabling comprehensive studies that interrogate all classes of genome variation, genome-wide, across the entire allele frequency spectrum. Ongoing projects worldwide are sequencing many thousands – and soon millions – of human genomes as part of various gene mapping studies, biobanking efforts, and clinical programs. However, while genome sequencing data production has become routine, genome analysis and interpretation remains a challenging endeavor with many limitations and caveats. Here, we review the current state of technologies for genetic variant discovery, genotyping and functional interpretation, and discuss the prospects for future advances. We focus on germline variants discovered by whole genome sequencing, genome-wide functional genomic approaches for predicting and measuring variant functional effects, and implications for studies of common and rare human disease.

Genome editing experiments are generating an increasing amount of targeted sequencing data with specific mutational patterns indicating the success of the experiments and genotypes of clonal cell lines. We present EdiTyper, a high-throughput command line tool specifically designed for analysis of sequencing data from polyclonal and monoclonal cell populations from CRISPR gene editing. It requires simple inputs of sequencing data and reference sequences, and provides comprehensive outputs including summary statistics, plots, and SAM/BAM alignments. Analysis of simulated data showed that EdiTyper is highly accurate for detection of both single nucleotide mutations and indels, robust to sequencing errors, as well as fast and scalable to large experimental batches. EdiTyper is available in github (https://github.com/LappalainenLab/edityper) under the MIT license.

Functional mechanisms remain unknown for most genetic loci associated to complex human traits and diseases. In this study, we first mapped trans-eQTLs in a data set of primary monocytes stimulated with LPS, and discovered that a risk variant for autoimmune disease, rs17622517 in an intron of C5ORF56, affects the expression of the transcription factor IRF1 20 kb away. The cis-regulatory effect on IRF1 is active under early immune stimulus, with a large number of trans-eQTL effects across the genome under late LPS response. Using CRISPRi silencing, we showed that the SNP locus indeed functions as an IRF1 enhancer with widespread transcriptional effects. Genome editing by CRISPR further indicated that rs17622517 is indeed a causal variant in this locus, and recapitulated the LPS-specific trans-eQTL signal. Our results suggest that this common genetic variant affects inter-individual response to immune stimuli via regulation of IRF1. For this autoimmune GWAS locus, our work provides evidence of the causal variant, demonstrates a condition-specific enhancer effect, identifies IRF1 as the likely causal gene in cis, and indicates that overactivation of the downstream immune-related pathway may be the cellular mechanism increasing disease risk. This work not only provides rare experimental validation of a master-regulatory trans-eQTL, but also demonstrates the power of eQTL mapping to build mechanistic hypotheses amenable for experimental follow-up using the CRISPR toolkit.

© 2020 American Association for the Advancement of Science. All rights reserved. INTRODUCTION: The human genome contains tens of thousands of rare (minor allele frequency 800 genomes matched with transcriptomes across 49 tissues, we were able to study RVs that underlie extreme changes in the transcriptome. To capture the diversity of these extreme changes, we developed and integrated approaches to identify expression, allele-specific expression, and alternative splicing outliers, and characterized the RV landscape underlying each outlier signal. We demonstrate that personal genome interpretation and RV discovery is enhanced by using these signals. This approach provides a new means to integrate a richer set of functional RVs into models of genetic burden, improve disease gene identification, and enable the delivery of precision genomics.

We present an assay to experimentally test the regulatory effects of genetic variants within transcripts using CRISPR/Cas9 followed by targeted sequencing. We applied the assay to 32 premature stop-gained variants across the genome and in two Mendelian disease genes, 33 putative causal variants of eQTLs, and 62 control variants in HEK293T cells, replicating a subset of variants in HeLa cells. We detected significant effects in the expected direction (in 60% of variants), demonstrating the ability of the assay to capture regulatory effects of eQTL variants and nonsense-mediated decay triggered by premature stop-gained variants. The results suggest a utility for validating transcript-level effects of genetic variants.

Chromosomal translocations play a fundamental role in the evolution and speciation of antelopes (Antilopinae, Bovidae), with several species exhibiting polymorphism for centric fusions. For the past 35 years, the San Diego Zoo Global (SDZG) captive population of Soemmerring's gazelles has revealed complex karyotypes resulting from chromosomal translocations with diploid numbers ranging from 34 to 39. Poor reproductive performance of this species in captivity and elevated mortality the first month of life (perinatal) has been attributed to this chromosomal dynamism. We have extended the studies of karyotypic variation in the SDZG Soemmerring's gazelle population and analyzed the effect of chromosomal and genetic variation upon perinatal mortality. Karyotypes from 149 captive Soemmerring's gazelles were evaluated revealing two unreported autosomal combinations, now constituting a total of 15 distinct karyotypes for the 3 Robertsonian centric fusions originally described for this population. Among SDZG founders, distinct chromosomal variation and nuclear and mitochondrial genetic structure were detected corresponding to the institution of origin of the founders. Low levels of genetic distance and nucleotide diversity among individuals, in addition to high relatedness values, suggested that outbreeding is less of a concern than inbreeding for maintaining a sustainable captive population. Finally, analysis of karyotypes of offspring born into the SDZG Soemmerring's gazelle herds, in conjunction with the maternal karyotype showed association of chromosomal makeup with perinatal mortality. This supports the importance of continuing cytogenetic screening efforts, particularly to evaluate the presence of deleterious chromosomal rearrangements in stillborns. Zoo Biol. XX:XX–XX, 2015. © 2015 Wiley Periodicals, Inc.

Chromosomal translocations play a fundamental role in the evolution and speciation of antelopes (Antilopinae, Bovidae), with several species exhibiting polymorphism for centric fusions. For the past 35 years, the San Diego Zoo Global (SDZG) captive population of Soemmerring's gazelles has revealed complex karyotypes resulting from chromosomal translocations with diploid numbers ranging from 34 to 39. Poor reproductive performance of this species in captivity and elevated mortality the first month of life (perinatal) has been attributed to this chromosomal dynamism. We have extended the studies of karyotypic variation in the SDZG Soemmerring's gazelle population and analyzed the effect of chromosomal and genetic variation upon perinatal mortality. Karyotypes from 149 captive Soemmerring's gazelles were evaluated revealing two unreported autosomal combinations, now constituting a total of 15 distinct karyotypes for the 3 Robertsonian centric fusions originally described for this population. Among SDZG founders, distinct chromosomal variation and nuclear and mitochondrial genetic structure were detected corresponding to the institution of origin of the founders. Low levels of genetic distance and nucleotide diversity among individuals, in addition to high relatedness values, suggested that outbreeding is less of a concern than inbreeding for maintaining a sustainable captive population. Finally, analysis of karyotypes of offspring born into the SDZG Soemmerring's gazelle herds, in conjunction with the maternal karyotype showed association of chromosomal makeup with perinatal mortality. This supports the importance of continuing cytogenetic screening efforts, particularly to evaluate the presence of deleterious chromosomal rearrangements in stillborns.

Expression quantitative trait locus (eQTL) analysis is an approach for identifying how genetic variation between individuals contributes to inter-individual differences in gene expression. In this SnapShot, we describe the basic principles of eQTL analysis and its applications to diverse molecular traits of the cell measured by genomics and proteomics assays.