Sequenom Technology Core

This core facility now offers two Sequenom MassArray high throughput technologies:

1. SNP multiplex genotyping, ideally suited for genotyping a few dozen to hundreds of SNPs in dozens to several thousand DNA samples.
2. Quantitative DNA Methylation Analysis using Sequenom's MassARRAY Epityper technology. It is ideal for investigating a few or several hundred regions over multiple samples. We now offer to run your prepared samples through the Sequenom (details here).

• This methodology is one of the cheapest and most error free technologies for high throughput SNP typing. It uses samples arrayed in 384 well plates and allows custom genotyping of SNPs within candidate genes or genomic intervals.
• The Sequenom MassARRAY system relies on MALDI-TOF (matrix assisted laser desorption/ionization-Time-of-Flight mass spectrometry). Mass spectrometry is used to identify alleles on the basis of mass and a dramatic increase in the number of genotypes that can be called in a day. Since allele calling depends on mass it does not required expensive labeled primers and is also far more reliable than other genotyping approaches (< 0.5% error rate). This means that spurious associations are far less likely.
• The Sequenom SPECTRODESIGNER software designs primers for genotyping in multiplex fashion. The "IPLEX" software allow for the design of assays for up to 40 SNPs at a time.
• Briefly, the technology involves PCR amplification of the region containing the SNP of interest, an optimized primer extension reaction to generate allele-specific DNA products, and chip-based mass spectrometry for separation and analysis of the DNA analytes. A single post-PCR primer extension reaction generates diagnostic products that, based on their unique mass values, allow discriminating between two alleles. The entire process has been designed for complete automation including assay development, PCR setup, post-PCR treatment, nanoliter transfer of diagnostic products onto silicon chips, serial reading of chip positions in the mass spectrometer, and final analytical interpretation.
• Routine quality control (QC) of data generated in Sequenom's high-throughput center is performed on a daily basis to identify data not meeting minimal QC criteria, and to organize further processing as needed to improve quality. Data review is facilitated by an internal web-based application that accesses and summarizes raw and software-interpreted data stored in a data warehouse. Results are initially evaluated according to the fraction of assay successes per plate. For genotyping plates, an assay fails if it does not meet minimal criteria of mass spectral quality as determined in real-time by the MassARRAY software. If greater than 10% of assays on a plate fail, then the plate fails QC. When a plate fails, visual analysis of spectra from these wells is used to determine reasons for failure. In addition to assay success rates, genotyping plates are reviewed for results from positive- and negative-DNA control wells that are organized in specific patterns to assist in the QC process and to ensure correct plate orientations during processing and data review.

1. Investigator provides core with SNPs or genes for analysis. In the latter case, tag SNPs, non-synonymous SNPs, regulatory SNPs will be selected as requested.
2. Investigator provides high quality genomic DNA diluted to 5 ng/μl. Alternatively, concentration determination and plate preparation is provided as an extra service by the core. DNA handling information may be found here. We often refer clients who need help preparing DNA samples from tissue or blood to the Psychiatry Molecular Genetic Core, directed by Petra Nowotny (email: nowotnyp at psychiatry.wustl.edu ).
3. Custom genotyping involves primer ordering, dilution, and PCR of samples in 384 well format. This is followed by sample clean-up, spotting onto chip, analysis with the MassARRAY system and genotype calling.
4. Results include genotypes with the sample codes and a summary of success rates. Results will be emailed to the investigator.

Ten 384-well plates can be spotted a day. A routine 20-plex reaction yields 76,000 genotypes per day. Hence the instrument has a formidable capacity and can handle all requirements of Washington University.

We are located on Scott Avenue in the Biotech building (across from Olin Hall), 3rd Floor, room 309.
For technical help, phone Shenghui Duan at 314-747-1057.
For questions about SNP designs, use the query form (if not Washington Univ, use this query form), or email Cindy Helms directly, address is chelms at genetics.wustl.edu

Shipping Address:

    Bowcock Laboratory, Attn: Shenghui Duan
    Washington University School of Medicine
    Dept. Genetics, Human Genetics, Campus Box 8232
    Biotech Bldg., Room 309
    4566 Scott Ave.
    St. Louis, MO 63110
   

Mailing Address:

    Bowcock Laboratory
    Campus Box 8232
    Washington University School of Medicine
    660 South Euclid Avenue
    St. Louis, MO 63110
   

Genotyping Service Costs: *** UPDATED October 28, 2010 ***

SNP genotyping costs are based on sample size and number of multiplexed markers (as low as $0.19 per genotype, see pricing table below). Prices include basic supplies and labor, excluding primer costs which are billed separately. WUSTL clients will be asked to supply us with a purchase order number for IDT, our preferred vender for primer orders. Price includes multiplex design and one round of redesign for the set. After that, each redesign will cost an additional $150.

Primer costs: We highly recommend that we do the multiplex primer designs and order the primers from our supplier (IDT). For local clients, we'll ask for a WUSTL PO number to use in the IDT order. Clients from outside Washington University will have the primer cost included in the genotyping invoice. When we order the primers, we get a volume discount of about $16 per SNP assay for projects with 24 or more markers, otherwise the cost is about $20 per assay. More primer is required for over 2000 samples. There is usually a $45 shipping charge.

DNA quantification costs are separate: Cost of NanoDrop Spectometer DNA quantification is $100 per hour, usually taking one hour for a 96-well plate. You can then use the DNA concentration to dilute and array into plates, or we can prepare dilutions and array the samples into plates at $100/hour. Because of limitations on our time, for $25 per hour, we encourage users of the genotyping core to come to our facility to determine DNA concentrations with our machine to make the most accurate dilutions (see below).

Genotyping costs: In our system, one multiplex may be used in each quadrant of a 384-spot matrix, and our charges are based on the number of quadrants your samples occupy. The lowest rate is attained with sets of 384 samples and 12 or more markers per multiplex ($0.19 per sample per marker). The Sequenom MassARRAY Design software is used to find compatible PCR and extension oligos for multiplexes. Unfortunately we cannot guarantee ahead of time how many of your markers will be designed into each multiplex. Your costs are based on number of markers in each multiplex and sample size. Please note that we have reduced prices for Washington University clients starting in July 2010. We have held the overall cost for non-Washington University clients to be roughly the same as in past years. This means there now is a 45% genotyping surcharge for non-WUSTL clients, corresponding to approximately $0.276 per genotype for non-WUSTL clients, when using the fullest genotyping configuration (>12 SNPs, 384 samples).

	Genotyping Cost for N Markers in Multiplex (WUSTL clients rate)
#Samples	1-12 Markers ($ minimum)	N = 13	N = 14	N = 15	N = 16	N = 17	N = 18	N = 19	N = 20	N = 21
1-96	$268	$274.56	$295.68	$316.80	$337.92	$359.04	$380.16	$401.28	$422.40	$443.52
97-192	$513	$524.16	$564.48	$604.80	$645.12	$685.44	$725.76	$766.08	$806.40	$846.72
193-288	$736	$748.80	$806.40	$864.00	$921.60	$979.20	$1036.80	$1094.40	$1152.00	$1209.60
289-384	$935	$948.48	$1021.44	$1094.40	$1167.36	$1240.32	$1313.28	$1386.24	$1459.20	$1532.16

	Genotyping Cost for N Markers in Multiplex
#Samples	N = 22	N = 23	N = 24	N = 25	N = 26	N = 27	N = 28	N = 29	N = 30	N = 31
1-96	$464.64	$485.76	$506.88	$528.00	$549.12	$570.24	$591.36	$612.48	$633.60	$654.72
97-192	$887.04	$927.36	$967.68	$1008.00	$1048.32	$1088.64	$1128.96	$1169.28	$1209.60	$1249.92
193-288	$1267.20	$1324.80	$1382.40	$1440.00	$1497.60	$1555.20	$1612.80	$1670.40	$1728.00	$1785.60
289-384	$1605.12	$1678.08	$1751.04	$1824.00	$1896.96	$1969.92	$2042.88	$2115.84	$2188.80	$2261.76

	Genotyping Cost for N Markers in Multiplex
#Samples	N = 32	N = 33	N = 34	N = 35	N = 36	N = 37	N = 38	N = 39	N = 40
1-96	$675.84	$696.96	$718.08	$739.20	$760.32	$781.44	$802.56	$823.68	$844.80
97-192	$1290.24	$1330.56	$1370.88	$1411.20	$1451.52	$1491.84	$1532.16	$1572.48	$1612.80
193-288	$1843.20	$1900.80	$1958.40	$2016.00	$2073.60	$2131.20	$2188.80	$2246.40	$2304.00
289-384	$2334.72	$2407.68	$2480.64	$2553.60	$2626.56	$2699.52	$2772.48	$2845.44	$2918.40

We do not prepare DNA, but recommend the Psychiatry Molecular Genetic Core, directed by Petra Nowotny (email: nowotnyp at psychiatry.wustl.edu ) for our clients who need DNA prepared from tissue or blood.

For genotyping with the Sequenom:

• 15 ng DNA is used per genotyping reaction (i.e., one multiplex).
• A good even level of PCR product is needed for high quality results.
• The concentration of your DNA should be determined as accurately as possible.
• A260/230 and A260/280 ratios of 1.8 indicate ideal DNA purity and are more likely to succeed.

1. Estimating DNA concentration on gels
   • This is the least accurate method to determine DNA concentration.
   • If you elect to use this method take the photo of your gel at the point at which your unknown and your standard bands are just about to disappear from the photo. This will give you the most accurate estimate for this method.
2. OD260/280
   • This method can use a large volume of DNA, depending upon the spectrophotometer that you use.
   • Be sure you are in the linear range of your spectrophotometer when you take your readings.
3. Pico Green (Molecular Probes, Eugene OR)
   • This a sensitive method for determining DNA concentration.
   • It uses a small amount of DNA.
   • It depends on the sensitivity of your detection equipment, as little as 2 μl of a 50 ng/μl solution, or a 10^-4 dilution of most DNA preps from blood.
4. Nanodrop Spectrophotometer
   • This has become our preferred method for DNA quantitation, as it does not require any dilution to determine sample concentration, so the reading is more accurate.
   • Only 1.0 μl each DNA sample is read directly, and appropriate dilution for the stock is calculated.
   If you want to use the Genotyping Core's Nanodrop Spectrophotometer to determine your DNA sample concentrations
   • The current charge for use of the machine is $25/hour. You can estimate 1 hour per 96-well plate of samples. First prepare your DNA samples at concentrations estimated to be close to 50 ng/ul, and bring a picture of 2 ul each DNA run in 1% agarose gels with an appropriate DNA marker. You will use 1 ul stock for the DNA concentration determination. Nanodrop Spectrometer readings are direct concentrations, from which you can easily determine the appropriate dilution for each sample.
   • You then provide us with the newly diluted DNA plate that we genotype.

• It is best to standardize your DNA samples to a single concentration.
• We recommend making a MASTER PLATE of DNAs arrayed in a 96 well format. This should be approximately 50-100X more concentrated than the working concentration for your genotyping application (usually 50ng per μl for human DNA). Make enough plates to allow you to complete your entire project. We array our concentrated DNA stocks in 500μl deep-well trays, 96 wells per tray, obtained from Midwest Scientific (P9606, $115).
• Store your original tube stocks of DNA in a different location than your master plates. This will give you a back up in case of a disaster.
• For Sequenom genotyping, prepare DNA WORKING PLATES from the MASTER PLATES. Use a working concentration of 5ng/μl for high quality human DNA samples such as those from Coriell Institute. Each multiplex uses 3 μl DNA per well or 15 ng per multiplex. The robotics also require a minimum 20μl dead volume for accurate pipetting, so add at least that amount to the total required for the number of multiplexes. Any unused DNA in the plates may be returned to you. 96-well plates with DNA samples for Sequenom genotyping should be low-profile plates with V-bottom (as opposed to U-bottom or flat-bottom) wells. We prefer to receive DNA in skirted or semi-skirted plates. Each DNA plate you prepare will need a sample sheet. See section 'Sample File Preparation' for an explanation.
• Working DNA trays can be stored at -20°C. Masters should be stored at -80°C.
• It is a good idea to give your DNA batch numbers; it may help track down a problem in the future.
• All of our charges are based on the number of 384 well plates that are needed to complete your project so we recommend that you fill the space with samples to optimize your costs. Ideally there should be a duplicate of at least one sample and one blank per plate for controls.

Before bringing your samples, print this sample-submit-form and bring the filled-out paper with your plate(s). Label the DNA plate (not the disposable cover!) with your (lab) name and date, along with any other identifiers needed to correlate it with your electronic sample sheet (templates below).

If you prefer to submit DNA dried in a 384-well plate for each multiplex (15 ng, ready for PCR), we require ABgene plates, catalog number TF-0384.

Electronic Sample File Preparation You will receive your genotype data files formatted with the sample names you give us in an electronic sample sheet. Use the appropriate template (below) for each plate you submit for genotyping, replacing the word 'blank' with the appropriate sample names. The template file lists well positions in the order we need. Name the file after your lab and the plate ID, (please no spaces in the name), save the file as a text file and email it to: sduan at genetics.wustl.edu .

• 96-well plate template file
• 384-well plate template file

Note:We have limited space to store DNA plates. If you want the remainder of the DNA returned to you after genotyping is completed, please pick up your plates within a week of receiving your genotyping results. We will be forced to dispose of DNA trays that are left with us for more than a month.

• Most SNP sequences are easily incorporated into Sequenom multiplex assay designs. Common reasons for design failure are: SNP falls in a duplicated genomic region, another SNP maps within extension primer region (~15 bp of targetted SNP), and extensive repetitive sequence flanking SNPs prevent unique primer choices.
• Custom SNPs. If you have new SNP discoveries that you want to type using the Sequenom, please see the section below about custom SNP sequence formatting
• Tag SNPs from the HapMap Project usually work well in the assay design phase.
• For association studies in a defined genomic region, we recommend using linkage disequilibrium (D' or r² statistics) to find tag SNPs. Two widely used programs are Haploview and Tagger. If you prefer we can choose tag SNPs for you (at $150/hour). See the information given on the query form here or email chelms at genetics.wustl.edu for details.
• For large genomic or chromosome linkage scans, you may want to choose two or three validated SNPs per cM to cover the genomic interval. We plan to post human sets of screening SNPs for genomic regions here soon.

Submitting Your SNP Choices for Sequenom Assay Design

• Custom SNPs. If you have new SNP discoveries that you want to type using the Sequenom, please see the section below about custom SNP sequence formatting.
• If you are not sending formatted sequence, we require a list of names (rs-numbers) for the SNPs you want to use. We will format them for the assay design.
• We can also work with a combination of the above.

• Note: Some SNPs fail the assay design process (10% on average). We'll need to know how you want to proceed if some SNP choices fail (you send alternatives, we choose replacements for you (based on D' or r²values with the failed SNP(s)), or if we should proceed without replacements for SNPs that fail the design).

If you have identified new SNPs (rather than known ones such as those with an 'rs' prefix), and wish us to include them in assay design please provide an ID and sequence in the following format, masking repeats and any other polymorphic alleles as 'N' or 'n'. Provide at least 70 bp of non-repetitive sequence flanking the SNP site. File format consists of a tab-delimited file with a specific header, followed by SNP name and sequence, one per line:

SNP_ID(TAB)Sequence
SNP1name(TAB)About70-200bpSequence[variant1/variant2]About70-200bpSequence
SNP2name(TAB)About70-200bpSequence[variant1/variant2]About70-200bpSequence 
etc.

(click here for a 3-SNP real sequence example)

Save your SNP sequence file as a text file (.txt) and email to: chelms at genetics.wustl.edu Alternatively, if given the variant and exact SNP position within a particular genomic build, we can extract the flanking sequences and do the appropriate masking and formatting.

You will receive several results files, all tab-delimited text files. The links below are to actual results for 8 human SNPs, where sample names and rsSNP names have been changed. Note that some browsers will wrap lines. To view text files properly, download and open with a text editor.
1. A summary report listing the marker genotype success rates.
2. If there are duplicate samples in the run, a duplicates file reports which samples were duplicates and gives details for discrepancies detected between duplicate genotypes. To have samples reported as duplicates, they must be listed with the exact same sample name in the electronic sample sheet(s).
3. Genotypes file (where discrepant duplicates are zeroed). Markers are listed across the top and samples down the left side. The genotypes assume all markers are autosomal and can be reported with base letters, or numerically, where A=1 C=2 G=3 T=4. Our default allele-delimiter (as shown) is a space, but you may request comma or slash delimiter.
4. Upon request, we can also supply individual marker files that include qualitative genotype information.