Clinical Genotyping Instrument Guide

Table of Contents

Letter from the Editor
Index of Experts
Clinical Genotyping Experts:
Robert Keefe
Taimour Langaee
Eastwood Leung
Colin Ross
Resources

Download the PDF version here

Letter from the editor

Genotyping is moving deeper and deeper into the clinic where it can be applied to predict disease susceptibility, pharmacogenomic studies, validate SNPs, and more. There's a lot pinned to being able to genotyping DNA quickly and accurately. But that ability — and any result — is only as good as your instrument.

There are myriad ways to go about your genotyping projects — how are you going to choose a platform? In this Instrument Focus Guide, our group of experts share what they considered when choosing a technology platform. Topping their lists are accuracy, reliability, and cost-effectiveness. After all what good would a platform be if you couldn't trust its results? They also consider what changes a new instrument may wreak on your workflow. Our group of experts is here to help you wade through all these concerns about choosing a clinical genotyping instrument.

— Ciara Curtin

Index of Experts

Genome Technology would like to thank the following contributors for taking the time to respond to the questions in this guide.

Robert Keefe
Applied Genomic Technologies Core
Wadsworth Center/New York State Department of Health

Taimour Langaee
Director, Center for Pharmacogenomics
Genotyping Core Laboratory
University of Florida

Eastwood Leung
Director, Genomics and Proteomics Core Laboratory
Texas Children's Cancer Center

Colin Ross
Genomics Coordinator, Canadian Pharmacogenomics
Network for Drug Safety Department of Medical Genetics
University of British Columbia

Robert Keefe
Wadsworth Center, Albany, NY

GT: What kinds of scientific questions does your genotyping platform enable you to ask?

RK: As a resource facility, our job is to facilitate the search for answers to scientific questions being posed by investigators who request our services. Since "genotyping" in general involves the use of many different techniques to characterize the genetic identity, sequence, or sequence copy number at a particular locus, the broader scientific questions that investigators try to answer are quite varied. We use our different instruments to genotype single nucleotide polymorphisms, short tandem repeated sequences (microsatellite regions), and sequence copy number, or to verify gene transcription. This is done to assist investigators in addressing such scientific questions as (to name a few): Do certain allelic profiles of individuals predict disease, or a predisposition or susceptibility to disease? Is it possible to discover genetic determinants in mosquitoes that allow them to host and transmit disease-causing viruses? Are cell lines or mouse strains that are obtained from different (commercial) sources — but identified as being derived from a single particular line or animal strain — genetically identical? Or, finally, can a speciation event be identified through the genotyping of organisms (e.g. insects) collected in the field over a large geographical area?

GT: How do you decide which platform to use?

RK: When we obtained the funding to purchase a few instruments for genotyping, we focused on those that represented the latest technology that also came with ready-to-use SOPs: we weren't interested in serving as beta testers for "cutting edge" technology, nor did we want to spend a lot of time developing or optimizing new applications for a new technology. We wanted relatively high sample throughput capability, but not so high that we wouldn't be able to keep the instrument busy, or that resulted in expensive reagents expiring before they could be used up.We estimated that instruments capable of genotyping up to 10,000 samples a week would suit our present and near-future needs (and this has proven to be the case).We settled upon a real-time PCR platform for SNP genotyping/gene expression/gene copy number applications (96- and 384-well) and capillary electrophoresis platforms (16-and 48- capillary) for carrying out what is esoterically known as "DNA fragment analysis." We use this latter platform for SNP, short tandem repeat (microsatellite), and gene copy number variation genotyping. We visited other core facilities where the instruments that we were considering were already in use (to get "real-life" feedback), and I obtained additional information from instrument users by attending meetings and interacting with contributors to the ABRF's electronic discussion group.

When choosing amongst our instruments for use in a new project, we select the one that provides accurate genotyping data in the shortest period of time and for the lowest cost. When discussing new projects with researchers, we first ask how many samples need to be genotyped, how soon the results are needed, and what the expense (to us and the investigator) will be. We then select a genotyping methodology based upon those answers.

GT: When shopping for a genotyping technology, what factors did you consider?

RK: We also ask whether the new technology will enable us to do current analyses moreefficiently or effectively. For example, will the new technology improve turn-around time and/or increase sample throughput without sacrificing accuracy? Can the technology realistically decrease our cost per sample/plate? If the technology allows us to provide a new type of genotyping analysis, is it being acquired to meet existing investigator needs? This latter question is best addressed by surveying the researchers at your institution to ascertain whether there is a current or projected need for a new instrument to warrant the expense of the purchase price and subsequent instrument maintenance costs. Is the technology relatively easy to learn and use, or will dedicated personnel be required to run it? How tested is the technology (what's its "track record"?), and what investments of time, effort, and money are needed to keep the instrument running optimally?

GT: Did you have to alter or establish a new workflow to accommodate your genotyping platform(s)?

RK: We didn't need to make large changes in adopting the genotyping platforms that we chose. We started out using vertical polyacrylamide gel electrophoresis instruments for genotyping work in 2000, and replaced them in 2003 and in 2006 with the capillary electrophoresis and real-time PCR instruments that I mentioned earlier. Recently, we added a pyrosequencing instrument to do short DNA sequencing and SNP genotyping work. Since all of the assays we use are DNA polymerase-based, we still maintain a general workflow of: sample reaction setup in a designated "clean" environment (pre-PCR); thermalcycling (on or off the genotyping instrument), with possible fluorescence-based detection of amplified product at this step; and possibly a third, post-PCR step (e.g. sample dilution) prior to fluorescence detection of (diluted) amplified product on our capillary electrophoresis instruments.

GT: How did you establish standards and, now, maintain your quality controls?

RK: The standards and QC guidelines needed or implemented are dependent upon the specific platform or assay used. Whenever possible, we try to obtain control samples from the investigator that will confirm all expected allelic outcomes for the genotyping analysis requested. Unfortunately, this is not always possible, for example, if a particular allele is extremely rare. In general, the QC measures that we implement will confirm that the instrumentation is performing optimally, the quality of the reagents used in our assays is good, and all primers and probes used in our PCR-based assays are targeting the correct genetic sequence(s). For example, we keep a panel of CEPH DNA samples on hand to test new TaqMan SNP primer/probe assays (including new assay lots). When we run the panel and observe the expected SNP genotypes, we confirm that our reagents are good, and that the probes are targeting the correct genetic locus. We also use other panels of DNA samples of known purity and concentration (purified and quantified by our staff) simply for testing reagent/assay quality and instrument performance.

GT: What training and technical support do your platforms require?

RK: All of our genotyping instruments required training for the new user. The training was provided to us shortly after instrument setup by vendor field engineers and was included in the instrument purchase price. After that, it's "learn by doing." Or, if additional training is needed at a later date, it is provided by the vendor at additional cost. I can't overemphasize how important it is to have actual data-generating experiments lined up for running on your new instrument during your training session(s). You and your staff will derive a lot more from the training experience by running actual samples. Also, your expertise will grow far more quickly if you continue to use the instrument regularly thereafter. It is difficult to know what questions to ask during training if you've never run a set of samples through the entire analytical process of the instrument. So, run some real experiments before the training engineer leaves town! It is also important to create (and update) good instrument SOPs: protocols that novices can pick up and use effectively without too much additional guidance. None of our current genotyping instruments has a service contract. Over the years, our technical support has come from: service contracts, free or fee-for-service phone and e-mail tech support; and networking with colleagues and other core managers at meetings, through direct correspondence, or through electronic discussion groups like that hosted by the ABRF.

Bob Keefe supervises the genotyping work carried out in the Applied Genomic Technologies Core at the Wadsworth Center in Albany, NY.

Taimour Langaee
University of Florida

GT: What kinds of scientific questions does your genotyping platform enable you to ask?

TL: To validate SNPs that were discovered through SNP discovery projects; to determine allele frequencies of novel SNPs and those reported without genotyping data; to determine if the SNP(s) of interest are associated with phenotype(s) in association studies; to quantify the alleles in allelic expression imbalance assays.

GT: How do you decide which platform to use?

TL: It depends on many factors such as: the type of genotyping assay (SNP, ID, or STR); the number of SNPs, and samples to be genotyped; the throughput, reliability, reproducibility, call rate. The cost-effectiveness, turn-around time.

GT: When shopping for a genotyping technology, what factors did you consider?

TL: Accuracy, reliability, reproducibility, throughput, cost of genotyping, cost of genotyping platform, advanced software, versatility (be able to do SNPs, IDs, and STRs, possibly), automation, technical support.

GT: Did you have to alter or establish a new workflow to accommodate your genotypingplatform(s)?

TL: We only worked to further automate the sample and genotyping processes and data conversion and transfer to the server.

GT: How did you establish standards and, now, maintain your quality controls?

TL: The standards in our core laboratory [are] based on the GLP, clinical, and laboratory standards, and molecular biology and genetic standard protocols for laboratory staff training, sample receiving, sample handling, processing, and archiving, DNA and RNA isolations and quantification, genotyping, data collection and analysis, and report output to assure quality, accuracy and reproducibility. Each genotyping platform usually offers some kind of standards and QC, and we also have our own in-house standards and QC, (blind genotyping, negative controls, positive controls, re-genotyping on different platform) as well as those that apply to different genotyping platforms.

GT: What training and technical support do your platforms require?

TL: Usually the training for each platform is provided and given by the technical support of the platform manufacturer. The training is naturally different for each platform and based on the genotyping method and throughput and can vary in time from one to three days. The need for technical supports varies from platform to platform, and depends on the experience and expertise of the staff.

Eastwood Leung
Texas Children's Cancer Center

GT: What kinds of scientific questions does your genotyping platform enable you to ask?

EL: Genome-wide association study of diseases, that is, the discovery of a genotype or a group of genotypes that are associated with the disease phenotype or clinical outcome, and copy number analysis of DNA samples.

GT: How do you decide which platform to use?

EL: The decision is based on the objective of the study and the budget of the project. For a genome-wide association study, one may want to start off with a high-density microarray-based platform. For detailed genotyping of a focused region/regions of DNA, sequencing platform is the choice. For multiplex genotyping of many samples, multiple platforms are available. These platforms include: low density microarray, and DNA polymerization together with fluorescence detection, denaturing high-performance liquid chromatography, capillary electrophoresis, or mass spectrometry.

GT: When shopping for a genotyping technology, what factors did you consider?

EL: We compare different platforms with the following parameters: reproducibility of the assay, the cost per genotype assay, the successful genotype call rate, the throughput (number of samples per day), and versatility of the platform.

GT: Did you have to alter or establish a new workflow to accommodate your genotyping platform(s)?

EL: Yes, each project may use a different setup of the platforms. Also, the platforms are constantly being updated in terms of throughput and density. Therefore, the routine workflow must be changed to accommodate the constantly updated protocol.

GT: How did you establish standards and, now, maintain your quality controls?

EL: In terms of genotyping, there are a series of first line parameters to check. These factors are reproducibility, overall signal intensity, and successful call rate.

GT: What training and technical support do your platforms require?

EL: Workflow can be familiarized after two rounds of hands-on experience. The companies always provide a Web-based manual, webinar, and phone-line technical support during working hours.

Colin Ross
University of British Columbia

GT: What kinds of scientific questions does your genotyping platform enable you to ask?

CR: Our genotyping platform primarily supports the genomic analyses of the Canadian Pharmacogenomics Network for Drug Safety, a large, Canada-wide (and growing) project that is focused on identifying genetic factors that contribute to severe adverse drug reactions. Two patients receiving the same dose of a medication may react very differently, and in some cases the adverse reaction will have very serious consequences. Adverse drug reactions are the fifth leading cause of death and are responsible for significant long-term disability. CPNDS clinicians and surveillance personnel from across Canada identify patients that have suffered severe ADRs, and drug-matched control samples, to collect biological samples and DNA. These valuable samples are genotyped to identify genetic variants that cause severe ADRs. CPNDS is then translating these findings back to the clinic for the benefit of patients. In addition to this work, the excess capacity of the platform allows us to support the genotyping needs of additional academic and commercial users in western Canada for a variety of research projects such as forestry health projects, family-based linkage studies, and large association studies.

GT: How do you decide which platform to use?

CR: The scale of a project primarily determines what platform we use, in terms of the number of genetic variants and the number of samples to assay. We primarily use an Illumina BeadXpress for smaller scale projects and clinical-grade genotyping, and an Illumina 500GX platform for larger research projects. Together, these platforms offer a variety of options to cover a wide range of genotyping requirement; from genotyping a single sample for one SNP, to scanning the genome of thousands of samples for over a million SNPs each.

GT: When shopping for a genotyping technology, what factors did you consider?

CR: Our first and foremost concerns were accuracy and reliability of the technology. Additional important factors were excellent technical support from the company, and of course cost is always important. Illumina has excelled in all of these requirements, and we have had an excellent ongoing relationship with Illumina.

GT: How did you establish standards and, now, maintain your quality controls?

CR: All of our new arrays
and tests are assayed on a standard set of at least 96 samples. All of Illumina's custom arrays and off-the-shelf products also have a large set of quality control standards integrated into every array, which we use for routine QC. In addition to this, we include sample replicates of known genotype and blanks in every run.

GT: What training and technical support do your platforms require?

CR: Training has been required whenever a new system is installed or a completely new type of assay is incorporated. Technical support has been required on rare occasions, although this can often be remedied over an Internet-linked connection to the equipment.

GT: Did you have to alter or establish a new workflow to accommodate your genotyping platform(s)?

CR: No.

Resources

As you get your clinical genotyping projects up and running, you're bound to have a few more questions. Here's a list of resources to help you get the most out of your genotyping platforms.

Publications

Fan JB, Oliphant A, Shen R, Kermani BG, Garcia F, Gunderson KL, Hansen M, Steemers F, Butler SL, Deloukas P, Galver L, Hunt S, McBride C, Bibikova M, Rubano T, Chen J, Wickham E, Doucet D, Chang W, Campbell D, Zhang B, Kruglyak S, Bentley D, Haas J, Rigault P, Zhou L, Stuelpnagel J, Chee MS. (2003) Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol. 68: 69-78.

Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV. (2006). Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes. Clinical Chemistry. 52: 494-503.

Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV. (2007) Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping. Clinical Chemistry. 53: 1544-1548.

Hansen LL, Madsen BE, Pedersen K, Wiuf C. (2007) Conflicting results in SNP genotype assessment. BioTechniques. 43 (6): 756-762.

Maresso K, Broeckel U. (2008) Genotyping platforms for mass-throughput genotyping with SNPs, including human genome-wide scans. Adv Genet. 60: 107-39.

Tsongalis GJ, Coleman WB. (2006). Clinical genotyping: The need for interrogation of single nucleotide polymorphisms and mutations in the clinical laboratory. Clinica Chimica Acta. 363(1-2): 127-137.

Shendure J, Mitra RD, Varma C, Church GM. (2004). Advanced Sequencing Technologies: Methods and Goals. Nature Reviews Genetics. 5: 335-344.

Weeks DE, Conley YP, Ferrell RE, Mah TS, Gorin MB. (2002). A Tale of Two Genotypes: Consistency between Two High-Throughput Genotyping Centers. Genome Research. 12: 430-435.

Conferences

AACC Annual Meeting
July 27-31; Washington, DC

AACR: Molecular Diagnostics in Cancer Therapeutic Development
September 22-25; Philadelphia, USA

Personal Genomes: Technology, Interpretation, and Challenges
October 9-12; Cold Spring Harbor, NY