Current cattle industry practices require that registered cattle have complete paternity and parentage determinations which can cost up to $40 per animal. Our proposed MGST method will have the ability to reduce that cost to just the several dollars range. By using the latest methods in massive sequencing provided by current sequencing devices we will be able to achieve this goal. A simple cocktail of small single strand DNA molecules (probes) directs the genotyping which is interrogated by a thermal stable DNA ligase which has excellent discrimination properties. Another common molecular biology method called the polymerase chain reaction then adds sample specific tags and other common sequences to the ligated probes. This creates a complex library of DNA molecules that now contain the samples genotype information. The library is then processed on a massive scale sequencing device which can sequence 40 million molecules. The data is then counted and these counts used to determine the samples genotypes for all the loci being tested. The method will permit up to 100 or more of genotypes per sample and 1000's of samples to be processed in parallel. It is this multiplexing which will dramatically drive down the prices of genotyping operations. While we will only test paternity and parentage in this project the technology can be applied to any DNA bearing organism and will be suitable for directed genotyping in humans, plants, and other commercially relevant organisms. OBJECTIVES: We have tested the feasibility of inexpensively genotyping cattle using an existing ligation based genotyping assay with high through-put next generation sequencing (NGS) technology. Our mass genotyping by sequencing technology (MGST) has the potential to be highly multiplexed in terms of the number of SNP positions to be typed as well as the total number of animals that can be combined in a single assay run. We anticipate that our results will demonstrate that genotyping by mass sequencing has the capacity to accommodate at least 96 SNPs and upwards of 3,000 animals per assay run. In our second probe panel that tests SNPs associated with parentage, paternity, and meat tenderness (PPTP), we have designed 113 probe sets and have completed the MGST tests, determining that at least 95 of the probe sets are able to accurately (>95% concordance with Illumina SNP50 genotype data) determine genotypes. Some SNP probe sets that had failed were corrected (8 of 18) by simply changing the strands that they hybridize to (top or bottom). Other probes present a high level of G:T mismatch error which cause the G signal to bleed into the T signal, producing clusters that are harder to differentiate. To alleviate this G:T mismatch error we have found that the placement of the universal base (deoxyinosine) needs to be optimized for each of these problematic probes. The placement of the deoxyinosine appears to be optimally placed between the 2nd and 7th 3' positions of the left hybridization sequence (LHS) probe. We will optimize the placement on all probes that appear to have a G:T mismatch error, this is being done currently and is expected to be completed within two months. The working PPTP still needs to be normalized so that each probe set produces a normalized signal output. This is done by raising and lowering the individual probe concentration in the hybridization reaction. The optimal number of PCR cycles and their effects on genotyping will be determined, with the expectation that lower PCR cycles will produce more normalized data sets. Normalized data sets will permit more samples to be included within a sequencing library. We have completed tests on a set of 100, 384, and 1536 animals using 96-well and 384well plates as required. We will test MGST on gDNA extraction techniques from hair follicle, dried blood spots, whole blood, and ear punches. We have successfully tested genomic DNA extracted using the KingFisher automated gDNA extraction system, which uses magnetic beads to capture genomic DNA from lysates. We will also test other magnetic beads DNA purification methods, as these methods can isolate a fixed amount of gDNA from a tissue lysate and may alleviate the need for the normalization of DNA concentrations. Our final test of the MGST will be on a collection of >3000 samples (this is the maximum number of reaction wells available on our PCR machines) within 5 months. These samples have been obtained from our USDA collaborators. APPROACH: Genotyping ligation-dependent PCR probes (n=113) have been designed which are fused to sequences that are required for next generation sequencing technology from Illumina. Genomic DNA samples supplied by our USDA collaborator will be mixed with the genotyping probe mixtures, hybridized overnight and then ligated with a temperature insensitive ligase. PCR with sample specific indexes will add barcodes to the ligation products after which all the PCR reactions will be combined and co-purified on silica matrix columns, in order to remove excess probes and PCR oligos. The complete library will be sequenced on the Illumina GAIIx platform in a single lane with a single end read of 36 bases and a second discrete barcode read to determine sample indexes. Raw data will be binned into sample ID, locus, and allele counts. This count data will be plotted for visual reference and scored for genotype using a dedicated custom calling pipeline. The called genotypes will be compared to the established USDA genotypes and concordance values established. Call rates will also be determined. This method permits at least 96 SNPs to be genotyped for a single gDNA sample and we have processed >1500 samples into a single library which is sequenced on a single GAIIx lane resulting in a considerable drop on the cost per SNP/animal combination. Probes that demonstrate confounding G:T mismatch effects will be reordered and the placement of the universal deoxyinosine base will be determined at positions 10 to 2 of the 3' end of the LHS. These tests are carried out using simple agarose gel resolved ligation-dependent PCR reaction and the ideal deoxyinosine placement can be determined with the naked eye. Genomic DNA extractions from blood, blood spots, hair follicles and ear punches, will be carried out following the directions of the gDNA isolation kit manufacturers. Extracted genomic DNA will be simply tested using an agarose gel resolved ligation-dependent PCR assay, and then in the full MGST. The genotypes determined using the MGST will be compared with genotypes previously obtained by the USDA using Illumina bovine SNP50 chips and the degree of genotype concordances determined. In addition the number of "no calls" will be determined and compared to the "no calls" of the SNP50 genotype data, where possible. This type of analysis will permit us the evaluate the MGST genotype data in context to the well validated array based genotyping platforms (SNP50 chip)
