Taqman SNP genotyping

How does the Source BioScience SNP genotyping contract research work?

Source BioScience need you to provide the SNP information for your project; either the surrounding sequence information with identified base change, or, specific Applied Biosystems SNP assay as detailed below. Source BioScience LifeSciences will obtain designed assays and test them for functionality. Validated assays will be genotyped on your DNA samples to provide data in an excel format.

 

What technology do you use for SNP genotyping?

Source BioScience LifeSciences uses the 5' nuclease assay chemistry with TaqMan® MGB probes for single-tube convenience and reliable performance. Data is collected on an ABI PRISM® 7900 HT Sequence Detection System.

 

How many SNPs can you genotype for me?

Source BioScience LifeSciences can provide genotyping for any number of SNP assays. Pricing structure will reflect the improved throughput seen with larger numbers of SNP assays and samples.

 

How many DNAs?

The nature of the 5' nuclease assay means that larger numbers of samples (ideally a minimum of 96) are required to enable clustering and scoring of data points. Typical project sizes are 750 to 3,000 samples for each assay. Liquid handling restraints mean that throughputs are optimised if DNA samples are provided as multiples of 384 with allowances for controls. Samples provided in this format will give the best value for money pricing.

 

What is your maximum throughput?

Our current capacity is in excess of 38,000 scored genotypes a day, dependent on numbers of samples per SNP assay.

 

What equipment do you use to obtain such a high throughput of genotyping?

Matrix PlateMate liquid handling robots capable of preparing in excess of 100 x 384 plates a day with low volume 5ul reactions; heat sealer; Duncan thermal cycler capable of amplifying ~150 x 384 plates in 2hrs; ABI7900 HT capable of reading 84 x 384 well plates in 3hours. Custom LIMS for sample and data tracking.

 

How does the 5´nuclease assay work?

In the 5' nuclease assay (also known as the TaqMan assay), allelic discrimination is based on the characteristic 5' to 3' exonuclease activity of Taq DNA polymerase. PCR using flanking primers is performed including fluorescent oligonucleotide probes in a homogeneous assay. The probes consist of a 5' reporter dye and a 3' quencher dye, and are specific to the region containing the base change in the region to be amplified. The 5' nuclease activity cleaves the probe if hybridisation occurs, releasing the reporter from the quencher. Two different probes with different fluorogenic reporters are put in the reaction for allele discrimination, one specific to complement each of the variant alleles to be typed. If there is a mismatch between the probe and target DNA sequence, the hybridisation is significantly reduced, therefore stopping the cleavage of reporter from quencher, and release of fluorescent signal. The amounts of each signal released indicate which allele(s) of the target region is present. The probes have been improved further by the introduction of non fluorescent quenchers, containing a minor groove binder in the probe, thereby increasing the specificity of the probe to the SNP region and increasing reaction standardisation and sensitivity.

FAQ taqman 1 allele_plot



Figure 1: Data from 5' nuclease assay reactions analysed on the ABI PRISM 7900HT Sequence detection System. The clusters of output of the fluorescent data are seen: only FAM signal, homozygous allele 1; only VIC signal, homozygous allele 2; increase in both FAM and VIC signal, heterozygous, both alleles present. The negative control is also shown.

 

How are SNP assays designed?

There are three methods of designing SNP assays for use on the ABI 7900HT:

  • Assay On Demand (AOD) - These assays, currently numbering over 200,000, are pre-validated assays available from ABI. We have used ~400 of these assays in the past and have found them to be of extremely high quality.
  • Assay By Design (ABD) - These assays are designed by ABI from the provided sequence information. These assays undergo a preliminary QC by ABI, and as a result only those assays passing QC will be billed. Under normal circumstances this is our first choice for designing new assays.
  • Manual Design - These assays are manually designed by Source BioScience LifeSciences following a strict set of guidelines. We recommend that this mode of design is used only for assays which fail ABD.

 

Can you type all SNPs including deletions/insertions?

All bi-allelic SNPs can be typed using this platform, including insertions/deletions; some more complicated assays eg (TT/GG) may require manual design.

 

How do I supply the SNP info for the ABD and Manual design?

Information regarding SNPs should be supplied to us in an Excel or tab delimited text file in the following electronic format:

SNP name

Base change

Sequence

Turnip SNP 1

A/C

......AGCTTA(A/C)GTAGCT......

Turnip SNP 2

T/C

......GCATA(T/C)GTCTNG......


The sequence should contain at least 150bp either side of the SNP. The SNP of interest should be placed within brackets eg (A/C). Any additional SNPs or uncertain bases within the sequence should also be highlighted as an N. Any additional information, for example TSC / RS numbers can also be included, but are not essential.

 

And what about specifying Assay On Demand SNPs?

Assay On Demand (AOD) SNPs need their specific assay ID. The assay ID as shown on the ABI web site should be supplied (eg C_2608211_1) Assays can be searched for using a number of criteria from the ABI online catalogue.

 

Can you design successful assays using the 5´ nuclease assay for every SNP?

Although we are able to design assays for virtually any SNP - we cannot guarantee their success. The success rate depends in part on the accuracy of the sequence information given. It is therefore essential that additional SNPs in the region of interest are highlighted. It should be expected that a few assays will fail ABIs ABD design criteria / QC, although in some instances this can be overcome through manual design. We expect that about 90% of bespoke assays (ABD and manual design) will be typable.

 

How do I specify my Sample information?

Sample information should be supplied in an Excel file, or tab delimited text file in the following format:

Plate name

Well reference

Sample name

Plate 1

A1

Turnip 142

Plate 1

A2

Turnip 143

 

 

What are the sample requirements?

DNA should be ~10ng/ul, with variation amongst the sample set limited as much as possible. We require 2ul of DNA for each reaction plus an additional 30ul excess to allow for robotic overages. Excess DNA can be returned at project completion. DNA samples should be supplied in 96 well format, as these plates will be used directly on our liquid handling robots it is suggested that you use one of the following plates:

  • Skirted Abgene thermo-fast plates (Cat#AB-0800) for volumes <150ul
  • Matrix 1ml blocks (Cat#4211)
  • Matrix 2D bar-coded track mate blocks (Cat#3711) for volumes >150ul

 

However we may be able to accommodate alternative skirted plates.



A single Non-Template Control (NTC) is required on each 96 well plate, this should be in the same well position - we suggest A1. We recommend duplicating a number of samples - for example the first /last column of each 96 well plate. This enables a measure of concordance to be generated, which will subsequently increase the confidence in the results generated.

 

How should I send the samples?

Samples should be sent on dry ice. Please refrain from sending samples on a Thursday or Friday.

 

Can you extract my DNAs if I have blood samples?

Yes, please contact us to find out more

 

What QCs do you carry out?

We test the quality of the assay and the quality of your DNA before starting to genotype in earnest. Manually designed assays are examined using a panel of 96 control DNAs: 47 duplicated DNAs and 2 negative controls. This enables us to measure the separation, clustering and reproducibility of the assay. Assays only pass this stage if there is no ambiguity in scoring and 100% reproducibility. It should be noted that rare SNPs can sometimes be difficult to QC because there are few observations of the rare allele. Your DNA will be tested using an assay that has previously been seen to function well, poor performance can highlight the need to improve your DNAs - especially important prior to starting a large project.

 

What if my assay doesn't work?

We will always tell you if your assays don't work. Assays that fail ABIs design criteria / QC can be manually re-designed, in some cases this assay may also fail our QC, these can be manually re-designed using the opposite strand (where possible).

 

What if my DNA doesn't work?

We will always tell you if your DNA doesn't work. If your samples fail our QC it may be possible to rectify the problem through additional dilution of the samples. We have found that in some cases it is not poor quality DNA that causes the problem, but high levels of impurities. However dilution of samples can also lead to increased scatter of the plots reducing the amount of data generated.

 

What impurities can impact on the quality of data?

BSA

will not inhibit Amplitaq

Ca2+

3.5mM or greater

CHELEX resin

resin will inhibit PCR - let resin settle before transferring extracted DNA sample

Chloroform

50mM or greater

Dimethylformamide

50mM or greater

DMSO

will not inhibit in low concentrations (1%)

DMSO

greater than 10% will inhibit Amplitaq

DTT

1mM or greater

EDTA

50mM or greater

Ferric ion

10uM or greater

Haemoglobin/heme

heme from porphorins will interfere with PCR

NaCl

50mM or greater

NH4

will not inhibit Amplitaq

NP40

will not inhibit Amplitaq

Phenol

50mM or greater

KCl

50mM or greater

Propidium iodide

will not inhibit Amplitaq

SDS

50mM or greater

Siliconized tubes

will inhibit Amplitaq

Spermidine

will not inhibit Amplitaq

Triton X-100

will inhibit Amplitaq

 

How long will I have to wait to receive my data?

It always takes between 1-2 weeks to receive the assays from ABI. Production genotyping will take about a week for small numbers of samples and SNPs (eg 384 DNAs x 10 SNPs); larger projects of hundreds of SNPs and hundreds or thousands of samples may take a several months. The start date for a project will depend on current schedule and receipt of assay.

 

How do you track my samples and data?

Source BioScience LifeSciences have an in-house developed laboratory information management system (LIMS) and a similarly developed SNP/primer tracking system.

 

What do I do next? / I still have questions

Contact us with your project details and timelines and we will prepare a proposal. Let us know how you would like to be contacted. Alternatively, we could arrange a visit or meeting.