Genotyping

Genotyping - Overview

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in the human genome. Any two individuals are predicted to vary at more than a million different SNPs scattered throughout the genome. A small fraction of this genetic variation is likely to explain the majority of the differences between individuals, including their predisposition to development of many common human diseases, such as cardiovascular disease, hypertension, diabetes, asthma, and cancer. A SNP is of greatest value for genetic mapping studies when both alleles have a frequency greater than ~20%. Some SNPs reside in open reading frames and lead to alternate amino acid sequences while others residing in regulatory regions may affect control.

Introduction

The Genotyping Division of the RPCI Microarray and Genomics Facility utilizes the Sequenom MassARRAY Compact and Illumina BeadStation systems to offer high-performance DNA analysis. The Sequenom platform measures the amount of genetic target material and variations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Maldi ToF MS) and is able to deliver reliable and specific data from complex biological samples and from trace amounts of genetic target material. The Illumina BeadStation system uses either the 96-sample Sentrix Array Matrix (SAM) or Sentrix BeadChips for 384- to 1536-plex custom targeted genotyping or whole-genome genotyping, respectively. Both the Sequenom and Illumina systems offer:

High-Performance SNP Genotyping
Allele Frequency Analysis
Gene Expression Analysis
Long-Range Haplotyping
Methylation Studies
Copy Number Verification

All applications are supported by the manufacturer's available hardware, software and proprietary consumables, which automatically generates analysis data and assigns a status level to samples.

The Genotyping facility employs state of the art equipment including Qiagen, Matrix and Tecan robotic pipetting devices, 5 x 384 well PTC-225 thermalcycling machines, a Samsung chip spotting device, and the MassARRAY compact system for determining the absolute mass of short DNA sequences. This high-tech equipment forms the core of the genotyping facility and enables throughputs in excess of 10,000 SNP genotypes per day. The Illumina system includes the BeadStation 500GX for Infinium and GoldenGate applications, two hybridization ovens and BeadStudio analytical software.

Our targeted genotyping service includes primer and assay design through to production of genotypes. All steps involved are highly automated and are tracked using a laboratory management system. In these method, multiplexed PCR and extension reactions are performed in a single well. Currently, multiplexing permits determination of up to 40 SNPs in a single well of a 384 well plate for the Sequenom system, and 384 to 1536 SNPs in a single well of a 96 well plate for Ilumina. Whole genome genotyping using the Illumina Infinium platform has the ability to genotype over 1 Million SNPs per sample depending on the array selected.

Project Design

Because of the unique characteristics of any genotyping project and the high quality we aim for, we are required to meet with each investigator who wishes to use our service. Deciding which platform to use is made jointly with the investigator after discussing project scope, size, aims, samples and budget. After choosing a platform, primer and assay design are carried out and a second meeting will occur to discuss the results. Depending on those results, we will provide a statement of work and proceed with the project or consider alternative genotyping methods as necessary. The assay design software will reject sequences that are considered problematic, but the user will have the option to include these at their discretion. We strongly recommend that the user include 5-10 % sample redundancy and a number of SNP-specific positive control DNAs in their sample plates to ensure proper calls. Genotype results are provided to the user in an electronic format with bioinformatics support from the facility.

SNP failures

All customers must be aware that not all SNPs submitted can produce successful assays. At the assay design stage, there is a drop-out rate of 10-15% of SNPs which fail assay design completely. This can be due to the sequence surrounding the SNP containing repeat regions, other SNPs in close proximity to the SNP of interest, database errors, AT rich regions and runs of nucleotides next to the SNP of interest.

If it is possible to design an assay, there is then a further chance of the assay not generating good quality genotype data. In the past, we have seen up to a 10% further loss of SNP assays at this stage. Only those SNP assays which we consider to produce reliable genotypes will be delivered to the customer and subsequently charged for, unless that data is specifically requested by the customer. With Sequenom, each SNP assay is validated on the Coriell SNP 500 DNA set to allow us to identify and remove poor quality assays before analyzing customer samples. This cost is associated with the oligo set up fee. The Illumina GoldenGate system does not allow for assay validation until after the oligonucleotide pools are synthesized and analyzed.

Our goal is to achieve > 97% success rate in genotype calls for a SNP, with a minimum cut-off of >95%. When lower success rates are achieved, it is the user’s choice as to whether to have the data.

SNP Analysis using Sequenom Mass Spectrometry

Targeted SNP Analysis using Sequenom Mass Spectrometry is best suited for genotyping 50s - 1000s of samples for 10s-100s of SNPs. All assays are performed in 384 well plates, so sample sizes (4 x 96 well, 8 x 96 well, etc.) that fill these plates are most efficiently assayed. Orders consisting of < 20 SNPs are more expensive per genotype than larger projects because smaller SNP sets usually lead to lower plexes which results in an increased cost per SNP-genotype. Thus, after the assay design stage, we provide a statement of work for the customer to allow him/her to decide which SNP assays should be run, providing information on the efficiency of the SNP pools designed, the number of samples and a per sample-genotype cost. Orders with low sample numbers with any number of SNP assays are possible using the Sequenom MassARRAY platform but they require special DNA plate layout to be cost effective. Contact us for details. The costs are dependent on the number of samples assayed and the number of SNP assays designed. Once a project begins, we will not accept additional SNPs into the existing assays. Any new SNPs can be included in a separate project with their own associated costs. Each project cost will be calculated on an individual basis, but the following breakdown by plate & SNP:

Plate and reagent cost: $550 for up to 40 SNPs on 380 samples (iPLEX Gold)

Oligo and set up fee: $75 / SNP

These fees are internal RPCI investigator rates, outside users please inquire about non-RPCI pricing.

To use the Sequenom Genotyping service, we require that you electronically submit DNA sample IDs (96-W plate format) and SNP flanking sequence (with rs or ss identifier). After acceptance, we require 100ng DNA dried down in 96-W plates, with two wells remaining empty for inclusion of facility QC positive and negative control samples. DNA must be eluted in water as TE may interfere with the genotyping assay. A260:280 of 1.7-2.0 is required for Sequenom genotyping unless the user agrees to assay these samples at their own risk. Users are responsible for providing documented, high-quality DNA for genotyping assays. Investigators can retain the labs services to quantitate the DNA after which time only samples meeting stringent QC will be genotyped.

SNP Analysis using Illumina GoldenGate

Targeted SNP Analysis using the Illumina GoldenGate platform is best suited for genotyping 96 to 1000s of samples for 384-1536 SNPs. All assays are performed in 96 well plates, so sample sizes that fill these plates are most efficiently assayed. After the assay design stage, we provide the customer the assay list to allow him/her to decide which SNP assays should be ordered. Once the number of DNA samples has been decided and before ordering, a statement of work detailing information on the OPAs/methods/materials and cost will provided. The OPAs designed for the GoldenGate system should all be ordered at once to minimize expensive set up fees.

Cost: The costs are dependent on the number of samples assayed and the number of OPAs designed. Each project cost will be calculated on an individual basis.

To use the GoldenGate Genotyping service, we require that you electronically submit DNA sample IDs (96-W plate format) and SNP flanking sequence (with rs or ss identifier). After acceptance, we require 250ng DNA dried down in 96-W plates, calculated using the picogreen assay (not a NanoDrop) with two wells remaining empty for inclusion of facility QC positive and negative control samples. A260:280 of 1.7-2.0 is required for GoldenGate genotyping unless the user agrees to assay these samples at their own risk. Users are responsible for providing documented, high-quality DNA for genotyping assays. Investigators can retain the labs services to quantitate the DNA after which time only samples meeting stringent QC will be genotyped.