Sample Number
Investigators are asked to attempt to have the total number of samples closely divisible by 82.
Data is generated in the laboratory in a 96-well plate format. We run 82 mouse samples on each
plate. We cannot combine mouse projects due to the specificity of the market set to each cross.
Please inform your CIDR contact of the exact number of experimental mouse DNAs you will
be sending.
Control DNA
For each 82 experimental samples, please send 10µg of DNA (400µl of 25ng/µl) for each of the
two parental strains in your cross and 20µg of DNA (2 tubes of 400µl of 25ng/µl) on a F1 animal
from the parents.
Blind Duplicates
We will need four blind duplicate samples (prepared by you) per 82 samples sent. Each blind
duplicate should be a replicate of another sample being sent to CIDR in that batch. As an
example, if you will be sending 164 experimental samples, you should plan on sending eight blind
duplicate samples (164 / 82 * 4). Choose your blind duplicates such that there are four from each
96-well plate. The key to break the code to identify the sample IDs for these blind duplicates
should be e-mailed to your CIDR contact before the samples are shipped. The information
needed to break the blind will be kept confidential until genotyping is completed. The sample amount
and concentration for each duplicate sample should be same as for an experimental sample and is
described in the Sample Preparation Section.
Sample Preparation
We require 10 µg of DNA per sample diluted to a concentration of 25 ng/µl in sterile water (i.e.,
400 µl of 25 ng/µl DNA for each mouse). After you have communicated to us the sample number
in your project, we will send you 96-well Matrix Deepwells, barcode labels, and instructions for
sample placement to prepare and aliquot your samples. Additional details about sample
preparation will be sent with these supplies.
Barcode Labels
We will generate and send you the barcode labels for your samples. Each label will contain the
CIDR ID in that identified the sample for us by project code (M+2 letter project specific code) and
well position. As you place your DNA samples into the labeled tubes you must keep a record of
your IDs for the animals and the corresponding CIDR ID. The genotyping data will be released to
you with only the CIDR ID.
Barcode labels will also be provided for the duplicate and control DNAs. Labels for the duplicates will be in a QC series (i.e. QC1-QCn). CIDR will reassign these QC samples with CIDR- compliant IDs after we receive them, thus preventing them from being identified as duplicate samples during the genotyping process.
- Quality Control
- • Four blind duplicate samples (prepared by you) will be run for each 82 experimental samples. Data from these will be used to calculate a project-specific error rate.
- • We will run two C57BL/6J controls, four parental strain controls (two provided by you and two provided by Jackson Laboratories), two F1 controls (provided by you), and two water blank PCR controls per 96 wells.
- • All allele calls will be examined for consistency with parental genotypes
- • Internal QC measures will be used to identify potentially problematic allele calls for additional review.
- • All data will undergo extensive post-genotyping analysis prior to release. This is done to assure that we have identified data generated due to lab-related errors. The post- genotyping analysis includes review of data point plots that represent the distribution and frequency of project alleles for each marker. These plots are provided to you as part of the data release.
- Project Goals
- • We strive for 90% completeness of all projects. This means that for the project as a whole, not more than 10% of the genotyping data will be missing. This goal is only applicable to samples of sufficiently good DNA quality that they amplify >75% of the time.
- • We also strive for a <1% genotyping error rate as measured by project specific blind duplicate samples provided to us.