The MCIC provides services for
- Capillary sequencing using the ABI 3100xl
- Genotyping: fragment analysis on Beckman CEQ8800, SNP analysis on Luminex200 or Illumina BeadXpress
- Deep genome sequencing using the Illumina Genome Analyzer II
Fragment analysis on the CEQ8800
Instrumentation
Instrument description and dyes:
Fragment analysis is run on the Beckman CEQ8800 instrument. This is an 8 capillary instrument with non proprietary software for DNA sequencing, STR (Short Tandem Repeat), SNP (Single Nucleotide Polymorfisms) analysis and AFLP (Amplified restriction Fragment Length Polymorphic) fingerprinting.
The CEQ8800 has diode lasers that excite infrared dyes, and has four detection channels. For fragment analysis, alleles in each sample can be labeled with up to three separate dyes, the fourth dye is used for the size standard. Currently Integrated DNA Technologies and Proligo are the only supplier of oligonucleotides labeled with infrared dyes (WellRED dyes). Three different WellRED dyes are available: D4-PA (650/670), D3-PA (685/706) and D2-PA(750/770). WellRED dyes have different signal strengths, therefore the signal strengths of amplification products will also depend on the dye employed for labeling. The Molar Extinction Coefficient of these dyes and subsequently their signal strengths are in order D4-PA>D3-PA>D2-PA. When pooling reaction, roughly equal signal strengths are obtained by adding the following relative volume of PCR reaction products (assuming the same amplification efficiency for all the alleles):
0.1 ul D4-PA PCR reaction;
0.2 ul D3-PA PCR reaction;
0.4 ul D2-PA PCR reaction.
Size standards:
Three different size standards are available:
CEQ DNA Size Standard Kit - 80 (P/N 608395): Contains two reference fragments labeled with the D1-PA dye for the use in sizing SNP fragments in the approximate size range 20 to 80 nts. The CEQ DNA Size Standard Kit - 80 is designed to accommodate a wide range of sizes for multiplexed and poolplexed SNP fragments.
CEQ DNA Size Standard Kit - 400 (P/N 608098): Includes DNA fragments of the following sizes labeled with D1-PA fluorescent dye: 60,70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, and 420 nucleotides. and is used for analysis of fragments up to 400 nucleotides
CEQ DNA Size Standard Kit - 600 (P/N608095): Includes DNA fragments of the following sizes labeled with CEQ WellRED fluorescent dye: 60,70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, and 640 nucleotides, and is used for analysis of fragments up to 600 nucleotides.
Fragment analysis software:
Genotyping data are analyzed with the CEQ800 Series Fragment Analysis Software that estimates DNA fragment sizes and amounts and identifies alleles represented by specific DNA fragments. This software runs on Windows2000 and WindowsXP professional. It requires SQL server capabilities, and administrator privileges to be installed and run. (WindowsXP Home Edition does not support SQL capabilities, therefore can not run the CEQ software). To install this software onto your computer, and to receive initial training, contact the MCIC staff. The CEQ 8000 Series Fragment Analysis Training Guide can be downloaded from this site. (download guide)
Instructions for sample submission for fragment analysis on the CEQ8800
Before starting a genotyping project you need to contact the MCIC staff, to discuss the project. This is necessary, because conditions for running each project need to be optimized. Similarly, when you change established genotyping conditions, or use new primers you need to let us know. To submit samples, you need to:
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Samples can be submitted in any microtiter plate. Facility staff will combine samples with formamide and size standards just prior loading them on the instrument. The amount of PCR product used will depend upon the efficiency of the PCR reaction. Beckman recommends the following dilutions (app.1/100 to 1/400):
0.1 ul D4 labeled sample (0.2ml for D3, and 0.4ml for D2)
0.5 ul size standard
40 ul loading solution (formamide)
Certain samples may require more or less volume of the PCR reaction be added. Adding more PCR reaction can reduce the signal strength of the size standard peaks and other sample that is co-injected. This effect is also exaggerated by salts in unpurified PCR reactions and/or unincorporated primers, because all negatively charged molecules compete in the electrokinetic injection. Optimal primer incorporation and salt removal may be required.
PCR reactions can be desalted as follows:
- add 0.25 ul of 20mg/ml Glycogen (Boeringer Mannhein, Cat.#901393) to the PCR reaction
- add 1/10 volume 3M NaAc pH 5.2
- add 2.5 volume 95% EtOH (from -20 degree C)
- centrifuge at max speed at 4 degree C for 15 minutes
- rinse the pellet twice with 70%EtOH (from -20 degree C); after each rinse, spin at max speed at 4 degree C for 2 minutes
- to completely remove the EtOH, invert the plate onto a paper towel and spin at 180 x g for a minute, then remove the plate from the centrifuge and let the pellet dry.
- resuspend in the appropriate amount of dd water.
How to retrieve fragment analysis data
You will be notified via e-mail, when your genotyping data are ready to be downloaded from the OARDC server. Follow the link http://www.oardc.ohio-state.edu/dnaseq , or download your data using a file transfer program ( FTP for PC users, Fetch for Mac users), with the following settings:
host: ftp.oardc.ohio-state.edu
user id : xxxxxxx
password: xxxxxx
User id and password are provided by the MCIC staff.
You are responsible to back up your data: the data will be deleted from the server after 30 days.
Rates for fragment analysis on the CEQ8800
The Beckman CEQ8800 is an 8 capillary instrument, therefore we charge per 8 sample, which represents one run. We provide the size standard and its cost is included in the run. For detail pricing please visit our "Rates" page.
Single Nucleotide Polymorphism (SNP) analysis on the Luminex200
Instrumentation
Instrument description:
The Luminex200 is a flexible analyzer based on the principles of flow cytometry.
The xMAP technology is a proprietary process to label polystyrene microspheres with two spectrally distinct fluorochromes. Using precise intensities of these fluorochromes, 100 different microsphere sets with specific spectral addresses are created. Each microsphere set can have a different reactant on its surface. Since each microsphere set can be distinguished by its spectral properties, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the molecular interaction that has occurred at the microsphere surface.
The samples are analyzed on the Luminex 200™ analyzer. Microspheres pass by two separate lasers in a rapidly flowing fluid stream, and the fluorescence of the microsphere is detected. High speed digital signal processing classifies the microspheres based on their spectral address and quantifies the reaction on the surface. Thousands of microspheres are measured per second resulting in an analysis system capable of analyzing and reporting up to 100 different reactions in a single reaction vessel in a few seconds. For more information on the instrument go to http://www.luminexcorp.com/products/luminex_200.html.
SNP analysis on the Luminex200 instrument.
For SNP analysis, internally fluorescently labeled microspheres (FlexMAP microspheres) are coupled with oligonucleotide sequences (anti- TAGs) that are an optimized isothermal set with minimal cross-reactivity. The second component of this assay is the user’s customized oligonucleotides, which must first be tagged with 24-base FlexMAP TAGs (see the list on the "Protocol for SNP analysis") in order to be captured onto the FlexMAP microspheres.
Several SNP detection assays can be used with the Luminex 200 with the FlexMAP microsphere, including
- Allele-Specific Primer Extension, (ASPE)
- Single Base Extension (SBE),
- Direct Hybridization (DH),
- and Oligonucleotide Ligation Assay (OLA) assay
For a comparison of these methods see: Lee et al. (2004). Comparison of four flow cytometric SNP detection assays and their use in plant improvement. TAG Theoretical and Applied Genetics. Volume 110:167-174.
ASPE was shown to produce robust results and to be cost-effective.
At the MCIC we optimized the protocol for ASPE assay and this is the method we are currently using. (download protocol)
Guidelines for sample submission for SNP analysis on the Luminex200
If you would like to use the Luminex200 please first contact the MCIC staff to discuss and the project. Below are the steps that you will need to do for the ASPE protocol:
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You will be notified via e-mail, when your genotyping data are ready to be downloaded from the OARDC server. Follow the link http://www.oardc.ohio-state.edu/dnaseq , or download your data using a file transfer program ( FTP for PC users, Fetch for Mac users), with the following settings:
host: ftp.oardc.ohio-state.edu
user id : xxxxxxx
password: xxxxxx
User id and password are provided by the MCIC staff.
You are responsible to back up your data: the data will be deleted from the server after 30 days.
Rates for SNP analysis on the Luminex200
We have two separate rates, which give you the flexibility to perform the ASPE extention in your laboratory. We charge for the microphere separately, as the amount that you will need for a particular assay depends on the number of samples and the number of genotypes to be analyzed. Please visit out "Rates" page for pricing details.