SNP Genotyping Takes Advantage of Faster Sequencing

SNP Analysis
Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.

Sometimes it is the small differences that cause the biggest problems. A single-nucleotide polymorphism (SNP) is a variation in a single nucleotide at the same locus in the genome between two individuals of the same species. Such a tiny genetic difference may have no consequence, especially if buried deep in a noncoding region of the DNA. Or sometimes one small difference of a single nucleotide can encode crucial, even lethal, information, such as the developmental or molecular switch for a devastating metabolic or neurological disease, marked resistance to contagions, or the luck to live cancer-free. SNPs are also valuable to researchers as biomarkers for diseases in genome-wide association studies (GWAS). Measuring the variations in SNPs among individuals in a group is the job of SNP genotyping. Recent strides in sequencing technologies are a boon to SNP researchers, and improvements in SNP genotyping technologies are likely just beginning.

Non-negotiable: sequence accuracy

A key ingredient for successful SNP genotyping is sequence accuracy. “The quality of the data provided by accurate and reliable SNP assays is the most important consideration for researchers – there is less ambiguity in the generated results, improving the SNP call rate over time,” says Handy Yowanto, global scientific marketing for Beckman Coulter Life Sciences. Beckman Coulter’s GenomeLab™ GeXP Genetic Analysis instrument, along with the GenomeLab SNPStart Primer Extension Kit, performs SNP genotyping by multiplexed single-base primer extension (SBE), using laser-induced fluorescence capillary electrophoresis. “The SBE technology uses four different fluorescent dye-labeled terminating nucleotides to interrogate the SNP allele at the 3' end of the target specific primers,” says Yowanto. “The primer extension approach, [a gold standard in the industry], takes advantage of the accuracy of the DNA polymerase.” The multiplex system allows researchers to analyze SNPs at multiple sites of a template or templates simultaneously.

Greater sensitivity is especially important when detecting rare mutations. Bio-Rad’s QX100™ Droplet Digital™ PCR (ddPCR) System, with its ability to measure the absolute number of target DNA molecules in a sample, is ideal for studying rare mutations. Its increased sensitivity and precision come from the partitioning of a standard 20 μl real-time PCR reaction into 20,000 nanoliter-sized droplets, followed by standard thermal cycling and droplet detection for amplification. Counting the PCR-positive and PCR-negative droplets (or partitions) gives an absolute measure of target quantity. Thus, the partitioning step is crucial to the QX100’s sensitivity. “Partitioning increases the local frequency of mutated DNA sequence relative to wild-type (or non-mutated) sequences, dramatically improving detection sensitivity and quantification of rare mutation sequences,” says Viresh Patel, marketing manager at the Digital Biology Center, Bio-Rad Laboratories.

The enhanced sensitivity and precision of ddPCR is valuable for analyzing different mutations. “Assay specificity is of the utmost importance for accurate and reliable genotyping,” says Patel, “particularly for mutation ‘hot-spots’ in the genome where multiple base substitutions can be found within a single codon. Digital PCR has significantly improved the sensitivity of mutation detection by 1 to 2 orders of magnitude relative to real-time PCR and sequencing.” Perhaps one day this sensitivity can be applied to difficult samples, such as crude or compromised DNA samples, which are currently challenging. “Non-amplification-based methods are less affected by sample quality, but also tend to be less sensitive for mutation detection,” Patel says. “A method that combines both benefits is ideal for broad applicability.”

High-throughput capacities: synergies between next gen sequencing and snp genotyping

Another recent leap in SNP genotyping technology is the advent of high-throughput capabilities, including systems that minimize care-taking by researchers. For example, Affymetrix’s Axiom® Genotyping Solution is designed to simplify the processing of results while requiring little operator intervention. Its Axiom® myDesign™ Genotyping Array Plates let you genotype as few as 480 samples, including proprietary markers, or markers from the Axiom Genotyping Database. “This comprehensive database [has more than 11 million] human variants, analytically verified for genotyping properties and minor allele frequency,” says Mary Schramke, senior director of strategic marketing for genotyping at Affymetrix. “The unique Axiom imputation data analysis workflow enables a higher genetic coverage, allowing samples to be processed more efficiently in follow-up GWAS. The high genotyping call accuracy allows researchers to customize fine mapping in a cost-effective way, to better characterize rare variants, reduce false positives, and maximize capture of population structural differences that are key to understanding the complete functional interpretation of genome sequences.”

Illumina capitalizes on the principles of parallelization in their microarray-based SNP genotyping system. Jennifer Stone, senior product manager at Illumina, thinks that their system’s main advantages are affordability, high throughput, and ease of use. “We offer the flexibility to genotype anything from a handful of variants to millions of variants per sample in the same experiment,” says Stone. Illumina’s system lets researchers target nearly any SNP in any species, using their standard or customized products.

Recently, DNA sequencing and SNP genotyping have been building on each other. “We’ve seen a synergistic feedback loop develop between sequencing discovery, and array-based SNP genotyping,” Stone notes. “Next-generation sequencing has enabled a tidal wave of discovery that has expanded the community’s catalog of known variants across many different species. Those newly-discovered variants are now providing exciting content for deployment on SNP genotyping microarrays, which can easily be used to genotype thousands of samples to boost statistical significance, and aid in pinpointing those variants of functional significance for a given phenotype or trait.”

Dennis Fantin, product management leader at Life Technologies, agrees that faster sequencing has, in turn, sped SNP genotyping. “The accessibility of rapid, benchtop sequencing provided, [for example,] by the Ion PGM™ Sequencer from Ion Torrent has facilitated the high-throughput screening of genes and gene panels from defined population samples,” says Fantin. “This has created a growing need to use our TaqMan® SNP Assays to confirm variants discovered during the screening process.” Life Technologies’ pre-optimized TaqMan SNP assays come in a user-friendly, single-tube format, as well as additional higher-throughput formats, including 96- or 384-well plates, TaqMan Array Micro Fluidic Cards, and OpenArray® Plates.

Generating corresponding functional SNP genotyping assays to these sequencing variants can be a time-consuming, multi-step process, however – which Life Technologies has simplified. “[We integrated] our Torrent Suite Software with the search portal for TaqMan Assays to enable direct submission of detected variants for further interrogation,” says Fantin. “In an effort to streamline workflows, mutations of interest can be selected in the Torrent Variant Caller Software. Over 4.5 million pre-designed TaqMan SNP Assays are available for germline mutations. The user can then select from the list of assays generated during the search, and move directly to online SNP genotyping assay ordering – a simpler way to move from sequencing results to SNP genotyping assays.”

SNP software

As sequencing technologies emerge, so does software evolve and emerge to take advantage of the new sequencing tools. “Affordable, easy-to-use software tools that support researchers’ ability to analyze the data at their disposal is critical,” says Schwei. “Although software often (and necessarily) lags a bit behind sequencing technologies, the gap has closed recently and powerful, easy-to-use software exists for SNP genotyping today.”

For example, Luminex’s popular xMAP® technology allows multiplexing of one hundred individual assays per reaction. “Designing a multiplex assay, however, has always been a researcher's nightmare,” says Abhimanyu Holkar, product manager for genomics at Premier Biosoft, which creates powerful software for scientists using SNP genotyping. Their PrimerPlex software simplifies primer design, a process that involves multiplexing millions of oligonucleotide combinations. “We also developed a multiplexing [software] solution for ligation-based high-throughput detection technologies such as DxTerity's Non-Enzymatic Amplification Technology (NEAT), and MRC-Holland's Multiplex ligation-dependent probe amplification (MLPA),” says Holkar. “PrimerPlex and AlleleID-NEAT™ are the only [software] offerings on the market for high-throughput multiplex SNP genotyping.” PrimerPlex also designs primers for multiplex allele-specific primer extension (ASPE) assays, a sequence-specific enzymatic assay for determining genotype.

Premier Biosoft’s Beacon Designer™ software is an oligo design tool for quantitative PCR (qPCR), and is meant to design primers for high resolution melt (HRM) analysis, a powerful and sensitive method of detecting SNPs and other genetic differences in double-stranded DNA. “For qPCR, Beacon Designer and AlleleID® are the most popular design tools,” Holkar says. “We are planning to develop software analyzing next-generation sequencing data for SNP genotyping application with better algorithms and data management solutions.”

DNAStar also supports large-scale SNP genotyping with software products. “Our SeqMan NGen® sequence assembly software performs ‘on the fly’ Bayesian statistical SNP analysis so that even the largest projects, like multiple human exome and genome data sets, can be assembled and analyzed quickly and efficiently,” says Schwei. “Our software can assemble billions of sequence reads and perform SNP genotyping on hundreds of samples often containing millions of variants, allowing the user to quickly identify the variants of interest.” 

DNAStar sees improvements in SNP genotyping workflows as one of the greatest needs in the market today. As a result, they are constantly improving their software, and regularly releasing product updates. “Recent improvements include the ability to compare SNPs across dozens or hundreds of genomic samples all the way from SNP identification, and comparison to common databases, through and including biological significance,” Schwei says. Besides improving their own products, software vendors must constantly monitor changes in sequencing technologies, as their software must incorporate new changes. “It is up to third-party software companies like DNAStar to continue to support the broader needs of life scientists,” says Schwei, “by supporting all sequencing technologies and workflows as they emerge and providing effective, affordable tools to life scientists to mine their genomic data for the most important information.”

Perhaps the most powerful application of SNP genotyping today is in disease research, which Yowanto believes is one of the most important advances in the field. “It opens up possibilities for researchers to better understand the prognosis of a disease or cancer,” he says, “and to study the development of better treatment options that could potentially improve patient outcomes in the future.” Databases that connect SNP genotypes to disease phenotypes are now helping physicians to use sequence information in treating patients. Genomic sequencing [is a powerful tool], says Schwei, but “much work remains in fully analyzing the genomic data and then, of course, translating that knowledge into biological significance and health ramifications.”

The image at the top of this page is Life Technologies' Ion PGM™ Sequencer.

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