Taqman SNP genotyping
How does the Source BioScience LifeSciences SNP genotyping
contract research work?
Source BioScience LifeSciences 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.
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 or go here
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.