The amplicons are 120–170 bp.
UMD 3.1 of Bos Taurus.
265 SNPs are targeted.
The assay uses 50ng DNA.
Commercial or lab-validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity must have an A260/A280 ratio of 1.8–2.0.
Use a fluorometric based method, such as Qubit or PicoGreen, to provide accurate quantification. UV-based methods, such as NanoDrop, measure any nucleotides in the sample, including RNA, dsDNA, ssDNA and free nucleotides, which can give an inaccurate measurement.
High-quality genomic DNA
The assay takes less than 8 hours total, with 2–3 hours of hands-on time. The libraries are then immediately compatible with the MiSeq System without any further manipulation.
One pair of oligos is designed for each amplicon. Hybridization of these oligos to unfragmented genomic DNA occurs in a 96‐well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. For additional details on the assay, see the assay reference guide.
Ideally, perform pre-PCR and post-PCR procedures in separate rooms. If separate equipment is used (eg, shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then the assay can be performed in designated, nonadjacent areas in the same room. Regular bleaching is outlined in the reference guide. In addition, filtered pipette tips are also recommended.
BVP1 is the amplicon oligo tube, which is the final pool of oligonucleotide probes used to generate amplicons. One BVP1 tube supports the amplification of all 265 amplicons in a TruSeq Bovine Parentage reaction.
An amplicon is a fragment of DNA produced by an amplification event such as PCR.
You do not need an upgraded MiSeq System to run these samples. However, consider the coverage needed for your study when designing an experiment. An online calculator is provided in DesignStudio to help with these calculations.
Set up a 1 x 121 run with dual indexing.
A manifest is required to sequence on the MiSeq System. The manifest file is available from MiSeq Support Downloads.
Use Sequence Genotyper to convert variant call files (VCF) into a genotype call file.