Research Genotyping

Price List

Marker List

STRP Genotyping

Project Information

Requirements for Samples:

DNA:

All samples should be diluted with filter sterilized distilled or deionized water to a concentration of 20-30 ng/µl. Do not use TE! The residual EDTA will negatively impact the small volume multiplexed PCR reactions that we perform for genotyping. If the dilutions of the samples are such that the residual EDTA concentration exceeds 0.05 mM we need to know that. The volume needed for your specific project will be communicated to you through email since the amount will vary by project depending upon the number of markers.
It is recommended that you retain some concentrated stock for each sample to use in subsequent studies and also in the event we require additional DNA to supplement weakly amplifying samples.
Please contact us before sending DNA acquired via buccal swab methods. We have had mixed results in the past with low amounts of buccal DNA. DNA obtained from the MDA reaction is an acceptable template for STRP analysis.
If needed we can provide DNA extraction and reagent protocols. Alternatively we can provide that service at PreventionGenetics along with Biobanking services.

Sample Tubes, Labeling and Shipping:

We will send you labeled tubes, racks, and plate seals for your samples after we receive your datafile (see below). To ship the tubes, we will need the address and phone number of a contact person.
The datafile entries should contain the kindred and individual number, Father ID, Mother ID and Gender. No patient identification information (such as names, social security numbers, or phenotypic status) should be provided. Kindred numbers and individual numbers should be kept as simple as possible. These two numbers will be used on the tube labels. Do NOT use any letters or hyphenated numbers in the kindred or individual identification numbers. Our programs do not accept alphanumeric character strings. If there is a unique lab ID that you would like included on the tubes please add that identifier in the LabID column on the spreadsheet. If provided that identifier will be included on the tube label. The datafile can be returned via email to dave.vaske@preventiongenetics.com.
When your samples are ready for shipping they should be frozen to -80oC. At that time also freeze enough ice packs to -80 degrees C to fill the Styrofoam shipping container. Once everything is frozen to -80 degrees C pack the samples and ice packs tightly into the container and send the shipment via overnight delivery to:

PreventionGenetics
Attn: David Vaske
3700 Downwind Drive
Marshfield, WI 54449
Phone: 715-387-0484

Requirements for Data Information:

Our programs will not accept alphanumeric, decimal or hyphenated numbers. There should be NO entries in the Individual column for samples that will not have DNA to be genotyped. Note also this file contains plate and well location for your samples. The returned file will be used for database and project set-up as well as to determine the number of samples for the final genotyping invoice.

It is asked that the following fields of information be completed within an Excel spreadsheet:

The kindred (family) number for each sample donor.
The individual number for each sample donor (required).
The individual numbers of both the father and mother of the sample donor if possible.
The gender of the sample donor, (1 for male and 2 for female; required).
Unique LabID, if desired and useful for your purposes.

An example of this data file is listed below:

Plate	Well	Kindred ID	Individual ID	Father ID	Mother ID	Gender
1	A01	1	302	0	0	1
1	A02	1	101	0	0	2
1	A03	1	402	302	101	1
1	A04	1	403	302	101	2
1	A05	1	404	302	101	1
1	A06	1	503	0	0	1
1	A07	1	511	402	503	1
1	A08	1	512	402	503	2
1	A09	1	513	402	503	1
1	A10	1	607	712	713	1
1	A11	1	612	607	403	1
1	A12	1	613	607	403	2
1	B01	1	615	607	403	2
1	B02	1	616	607	403	1
1	B03	1	617	607	403	1

Gender information will be used to identify any sample mislabeling through typing of X and Y chromosome polymorphisms. We recommend that all samples that come into the lab for genotyping have at least one gender specific marker typed with them! Family structures are used to search for violation of Mendel's segregation rules as a final check of the genotyping results. If your project is such that family structure does not exist, make certain each individual has a unique ID and leave the father and mother cells empty.

Currently, the maximum number of individuals for any one family is 86. If you have a family that is larger than 86 individuals, you will need to break the family down into two or more branches. Please give each branch a different family number. Family members need to be contiguous on a plate unless the sample number and start location for a family causes that family to get split across adjacent plates.

Genotyping Results:

The full address including email address and phone number of the individual who will receive the genotyping results should be sent to PreventionGenetics at the time of sample submission. Upon project completion, initial genotyping data will be sent via email to this individual. Once completed the final reports will be cut to a CD and sent via mail to the PI. Genotyping data will also be cut to the CD.

Genotyping results will be reported to you as allele sizes of the polymorphic markers in nucleotides. Allele sizes for all test samples are determined relative to the allele sizes assigned to the parents of CEPH family 1331 (133101 and 133102). Amplified DNA from these two individuals is used as standards on all of our gels. Reference genotypes for these two individuals for most markers may be obtained from the Center for Medical Genetics web site http://research.marshfieldclinic.org/genetics. Note that there may be differences between the sizes assigned to alleles and the actual physical lengths of the amplified fragments. However, in nearly all cases these differences will be no more than a few nucleotides. Note also that alleles with the same electrophoretic mobility will occasionally differ in sequence. For example, an allele with 18 dinucleotide repeats with structure (AC)10(TC)8 will be indistinguishable from an allele with (AC)8(TC)10 structure.

Genotyping results will be reported by chromosome and physical marker location along the chromosome. Thus a genome scan will have 26 excel files with genotyping data. Fine mapping projects will vary in file number depending upon the number of regions included in the project.

In the genotyping data files each marker is allotted four columns. These columns contain from left to right: allele 1 (larger allele in heterozygotes), allele 2, quality (either 0.99 or 0.90), and flag or indicator. The five indicators are:

' * ' - for putative mutation (violation of Mendel's rules given the family structure which you provided)
' &' - three or more alleles,
' $ ' -
' @ ' - gender error
' N ' - non-integer alleles (alleles which differ in length from other frequent alleles by a value other than an integer multiple of the repeat length).

Genotyping Quality:

Quality values (also known as confidence values) of 0.90 or 0.99 will be attached to each genotype for which we can determine alleles. Most genotypes will have quality values of 0.99; a few will be assigned the 0.90 values. The quality values are crude estimates of the probability that each genotype is correct. Analysts will have the choice of choosing quality value cutoffs for the data. For example, analysis programs may be run both excluding and including the 0.90 quality value genotypes. Goals for our large-scale genotyping efforts are > 95% average completeness of the genotyping data and < 1%genotype errors.

Completeness and quality of the genotyping data is highly dependent upon the DNA sample quality. If DNA samples are at the wrong concentration or are contaminated with materials which interfere with PCR, these samples will likely give poor results. We will identify samples which consistently amplified poorly at the end of the project.