Attachment 2 - WHB Genetic Analysis_Synopsis Specifications-Draft.docx

Genetic Analysis of Wild Horse and Burro Hair Follicle Samples Background The Bureau of Land Management (BLM) protects and manages wild horses and burros (WHB) under the authority of the Wild Free-Roaming Horses and Burros Act of 1971 to ensure that healthy herds thrive on healthy rangelands. The BLM monitors herds of free-roaming WHB on public lands. As part of that monitoring activity, BLM’s WHB Program monitors elements of genetic diversity in managed herds. This monitoring relies on genetic analysis of DNA from sets of samples of hair follicles taken from WHB that have been gathered from the wild. For the genetic analysis to be useful to BLM, it should be consistent with previous analyses. Specific measures and aspects of analysis for sampled herds are detailed in the synopsis specifications.

The BLM Wild Horse and Burro Program seeks to procure services that include the genotyping and analysis of hair follicle samples collected as a routine part of wild horse and burro genetic monitoring. Here, the determination of the allele lengths for an individual horse’s (or burro’s) hair, at a number of identified microsatellite loci (and, potentially, at other loci), is termed “genotyping.” The analysis sought is to characterize a number of attributes from each set of samples collected, including: observed heterozygosity, similarity to domestic horse (or burro) breed types, similarity to other BLM-managed herds, inbreeding coefficient, number of alleles identified per locus, and presence of any alleles not found in other feral horse samples. BLM is seeking sources that can both conduct the genotyping and produce the analyses for each set of genetic samples. The minimum purchase quantity will be 500 units of individual sample genotyping, 20 analyses, and 5 data transfers for a three (3) year period with potential option periods which could include additional sample genotyping, analyses, and data transfers.

Specifications/Performance Work Statement BLM sends the contractor samples from individual horses (or burros), in the form of hair and hair follicles that have been pulled from the horse (or burro) mane. Approximately 40 hairs per individual horse or burro are sent, with hair samples from a single individual enclosed in a dry envelope. Samples are labeled by BLM, and samples and explanatory data forms are sent to the contractor. BLM sends samples along with a data sheet that identifies sampled individual horses (or burros) by sex. Sets of samples usually include at least 25 samples per area being monitored.

The genotyping must be done in such a way that provides reliable characterization of microsatellite allele length for both copies of each sampled individual’s DNA. Currently accepted methods include but are not necessarily limited to using an automated DNA sequencer to separate polymerase chain reaction (PCR) products. Each individual’s DNA must be genotyped at, at the least, 12 microsatellite loci. Data about each individual’s genotype must be stored in a database, and those results must be shared with and periodically sent to BLM.

Qualifications:

The genotyping laboratory must have certification from the International Society for Animal Genetics (ISAG) at a high enough level to distinguish horse breeds, or be able to produce evidence that error rates for microsatellite genotyping are comparable to or better than those required for that type of ISAG certification.

Requirements:

I. Genotyping The contractor shall:

a) Genotype each individual horse’s (or burro’s) DNA (hair follicle) sample at enough loci to be able to characterize the sample set for analysis. The loci to be used in genotyping should include, but do not need to be limited to, the 12 microsatellite loci used to create the matrix of Fst values in Appendix F of the National Academies of Sciences 2013 report, “Using science to improve the BLM wild horse and burro program.” These loci include VHL20, HTG4, AHT4, HMS7, AHT5, HMS6, ASB2, HTG10, HMS3, ASB17, ASB23, and LEX33. Additional loci that the contractor may choose to genotype are optional;

b) Conduct genotyping with error rates for genotyping at a low enough level that allows for the contractor’s continuing accreditation by the International Society for Animal Genetics (ISAG);

c) In the even that a sample does not amplify clearly enough to distinguish a homozygote from a case of allelic dropout, the laboratory must have procedures in place to repeat the sample genotyping until a reliable result is obtained for that sample (or until it can be concluded that the DNA in the sample is of inadequate quality for amplification).

II. Data transfers and the database

a) For each sample, both copies of length polymorphisms at each locus shall be recorded in a database in such a way that meets ISAG standardization (i.e., so that the allele length labeled “M” is for the polymorphism that ISAG has identified as being in the middle of an identified range of polymorphic lengths for horse [or burro]);

b) The database shall contain records of all genotyped samples, where each sample is also associated with the animal’s identification number provided by BLM on the initial data sheet, sex, geographic source (including state, and herd management area), and date of sample collection (i.e., month and year);

c) The database shall be made using some form of database or spreadsheet computer software that is commonly available and widely used in contemporary American society;

d) The contractor shall send BLM a copy of the database with the results of BLM samples’ genotypes and all associated data at least once per every four analyses that are conducted, and also when the last analysis under this contract is conducted.

III. Analyses The contractor shall:

a) Briefly describe the number of samples received, the geographic source of the samples, the date they were received, and the laboratory and analytical methods used;

b) Include a table listing the genotypes for all sampled individuals, where each of the 2 microsatellite alleles for each genotyped locus is listed as a letter-based length polymorphism code, in keeping with ISAG standards;

c) Include a table showing, for each set of samples, the frequency of each observed allele, at each of the 12 loci genotyped;

d) Present a table with observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient (Fis), the effective number of alleles (Ae), the mean number of alleles per locus (MNA), the total number of variants (TNV), and the percentage of rare alleles (found at less than 5% frequency; RA). This table should also include the average, standard deviation, minimum, and maximum of the same measures for average domestic horses (or burros), and feral horses (or burros);

e) Identify whether or not the set of samples contains any unique alleles, not known from other feral or domestic horse samples;

f) Compare estimates of the measures in (b) from the currently analyzed sample set, against available measures from previous sample set(s) from the same area;

g) Present a table with quantitative measures of similarity to common domestic horse (or burro) breed types, including those of New World Iberian ancestry;

h) Present a dendrogram or comparable depiction of quantitative measures of similarity to established domestic breeds;

i) Present a dendrogram or comparable graphical depiction of quantitative measures of similarity to other BLM-managed herds;

j) Not provide management prescriptions or personal commentary about what the contractor thinks BLM should or should not do in terms of wild horse and burro management;

k) Prepare the analysis as a written report in pdf format, deliverable to a designated BLM employee and office, no more than one year from the date after receipt of the sample set.

IV. Data Ownership BLM shall be the owner of the intellectual property represented in the raw genotyping data from BLM-managed wild horses and burros. The contractor shall not publish or share genotyping results, database records of genotyped results, or analyses without prior written permission from the BLM Wild Horse and Burro Program Chief. The contractor shall not share or publish separate analyses of BLM wild horse and burro genotype results without prior written permission from the BLM Wild Horse and Burro Program Chief.