James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Human genetic variation is present in many different forms in the genome, including somatic mutations, single nucleotide polymorphisms (SNPs), copy number variations (CNVs) and structural changes (indels). Studying and understanding the variations takes a combination of powerful genotyping tools. This article focuses on two categories of tools which provide efficient workflows and enable researchers to gather more detailed information in their genotyping experiments.
PCR-based genotyping assay
“QIAGEN provides integrated sample-to-insight workflow for analyzing genetic variants via next-generation sequencing,” says Vikram Devgan, head of the biological research content business at QIAGEN. The company’s GeneRead DNAseq Targeted Panels V2 use multiplex, PCR-based target-enrichment technology; each panel is specifically designed to enrich for the most relevant genes and variants, delivering insight into cancer genetics. The panels also can be customized to include genes or other genomic regions tailored to customer-specific next-generation sequencing (NGS) project needs.
In addition, the panels can be used with any sequencer and sample type, including formalin-fixed paraffin-embedded tissue, liquid-biopsy and single-cell samples. The target enrichment takes three hours, with the entire workflow concluding in just four days. Data analysis is accomplished through Biomedical Genomics Workbench (a QIAGEN software platform). “With its broad selection of end-to-end analysis workflows, tools and visualization modules, it enables easy and accurate conversion of FASTQ to VCF files, empowering discovery, verification and validation of novel disease biomarkers,” says Devgan.
The LightCycler 2.0 real-time PCR instrument offered by Roche has been developed for detection of SNPs. With a run time of 30 minutes or less, assays can be performed rapidly in glass capillaries. Other advantages include high-level multiplexing, which enables the researcher to screen multiple genotypes simultaneously. Another PCR-based method, which was introduced in 2014, is the AriaMx System from Agilent Technologies. This real-time PCR instrument combines a state-of-the art thermal cycler, an advanced optical system with an LED excitation source, and complete data analysis software. Another advantage of using this instrument over others is that the detection system measures both SYBR Green and fluorogenic TaqMan probes, which provides flexibility in assay design to the researcher and enhances detection.
Thermo Fisher Scientific offers the OpenArray platform, which is a high-throughput, real-time PCR system for the profiling, confirmation, and screening of genotypes. This system offers several advantages, including speed, high throughput capabilities as well as economical savings. The throughput of the system can screen over 5,184 samples per workday and has the ability to generate over 110,000 genotypes per workday without using robotics. Economical savings can also be observed due to the miniaturized assay format reducing reagent consumption and also preserving precious samples.
Array-based genotyping tools
Illumina also has several tools for genotyping applications. The Infinium Assay, for example, is an array-based system in which genomic markers are interrogated using a two-step detection process. “Carefully designed 50-mer probes selectively hybridize to the loci of interest, with marker specificity conferred by enzymatic, single-base extension with a labeled nucleotide,” says John Picuri, product manager at Illumina. Next, detection of dual-color fluorescent staining of the labeled nucleotide is accomplished through the use of Illumina's HiScan or iScan imaging systems, which identify both color and signal intensity. For genotyping assays, the red and green signals specify each allele; homozygotes are indicated by red/red or green/green signals, and heterozygotes are indicated by red/green signals. For methylation, the color specifies the methylation status of the CpG site. For software analysis, Illumina offers GenomeStudio, Beeline and Assay Design Tool to facilitate analysis of microarray data and enable design of custom arrays.
When a scalable, high-throughput genotyping solution is needed, researchers might want to consider the Axiom® platform from Affymetrix. The Axiom 2.0 assay is a microarray-based genotyping assay that interrogates a SNP loci, indel breakpoint or CNV. The Affymetrix Axiom genotyping technology supports a range of human-based genomic applications, including but not limited to population genetics, prospective epidemiology studies in large biobank cohorts, genome-wide association studies, candidate SNP replication studies, pharmacogenomics profiling, human leukocyte antigen (HLA)¬ typing, transplant genomics and clinical-trial patient stratification. “Axiom® Genotyping Solution offers a uniquely flexible choice of innovative, pre-designed arrays covering more populations than any other technology,” says Marijo Gallina, strategic marketing manager at Affymetrix. The optimized design of these arrays enables combined genome-wide association studies (GWAS), replication and fine mapping, all in one study. “Axiom Genotyping Solution pre-designed arrays deliver cost-efficient discovery power with the most recent SNP content,” Gallina says.
To customize these arrays, the researcher might want to consider leveraging Affymetrix bioinformatics expertise to create an entirely new array for GWAS, replication or fine-mapping studies. “Custom arrays are created using our novel modular content approach to simplify and accelerate design customization,” Gallina explains. “These pre-designed content modules harness the power of the Axiom Genomic Database, a database of over 9.4 million variants and growing.”
These content modules are a main feature of the Axiom technology, which, in addition to offering cost-effective customization, simplify the process of designing arrays that perfectly match research interests. “Axiom modules are organized into high-value categories, including: population-specific genome-wide association study grids, loss-of-function markers, immune function and pharmacogenomic variants, enabling researchers to easily select curated sets of relevant markers.”
One limitation of this system is that all variants included on an array require prior knowledge for probe design, so only known variants can be detected. “Nevertheless, this limitation is minimized by large sequencing initiatives such as the 1000 Genomes project, which have identified the vast majority of human variants. These data continue to increase the genomic coverage and utility of arrays for most applications.”
On the software side, the Axiom Analysis Suite is recommended for use with the Axiom microarray platform. “The software is user-friendly with an intuitive graphic user interface designed to allow the user to set the desired settings and process through all genotyping steps with few clicks,” says Gallina.
Understanding genetic variation
Genetic variation can be sorted out through the use of several categories of genotyping tools. The PCR-based methods offer the option to amplify target genes, which can increase sensitivity of the assay. In addition, fluorescence in the assay format enables differentiation between homozygotes and heterozygotes. This feature can provide powerful insights, especially in tumor gene screens. Microarray-based genotyping tools, in contrast, offer a wider genomic variety in terms of loci probed. These arrays are also very customizable and offer a wide range of designs. Researchers have options for choosing either methodology or a combination of both, including both reagents and software analysis tools to carry out their genotyping experiments.