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MHC GENOTYPING
Genomics Services provides high-throughput MHC genotyping for investigators using nonhuman primates to study cellular immunity and transplantation tolerance.
The MHC region of macaques is much more complex than the human MHC, with each animal expressing a variable number of MHC class I alleles. To distribute animals appropriately depending on experimental goals, we sequence the most polymorphic segments of MHC genes or full-length transcripts, then group sequences into simplified haplotypes to indicate highly transcribed alleles. These haplotype designations make it possible to identify animals that share multiple MHC alleles. Standard genotyping reports include the full list of detected alleles, in addition to the simplified haplotype designations.
To balance experimental and control groups or determine the degree of MHC mismatch for transplant studies, lineage-level allelic resolution provided by our standard MiSeq assay (that focuses on the most polymorphic segments of MHC genes) is appropriate. When full resolution of the specific MHC allelic variant is important to discovery, we recommend PacBio SMRT sequencing of full-length MHC transcripts. If you are unsure of which MHC assay is best for your research, please contact Genomics Services.
For more information on our approach to MHC genotyping, please see our MHC white paper (pdf).
To request service, please complete the on-line WNPRC Genomics Services Service Request form.
OTHER IMMUNE LOCI GENOTYPING
Many investigators studying cellular immunity in nonhuman primates are interested in loci beyond the MHC. We offer the following genotyping services:
- TRIM5 genotyping via a MiSeq short amplicon sequence-based assay to detect TRIM5Q, TRIM5TFP, and TRIM5CypA alleles.
- Fc gamma receptor (FcɣR) genotyping via PacBio Sequel full-length sequencing
- Killer immunoglobulin-like receptor (KIR) genotyping via PacBio Sequel full-length sequencing
Though FcɣR and KIR assays are still under development for most nonhuman primate populations, we work with investigators interested in prototyping these assays as “early access” clients.
For more information including current pricing, please see our Other Immune Loci white paper (pdf).
Interested in more information? Contact the Genomics Services Host Genotyping group.
VIRAL PATHOGEN SEQUENCING
We specialize in RNA viral genome sequencing with an emphasis on simian immunodeficiency viruses (e.g., SIVmac239, SIVmac251, and SIVsmE660) and emerging pathogens (e.g., Zika virus). We use Oxford Nanopore Technology long read sequencing and Illumina MiSeq short read sequencing methods to sequence the entire coding sequence of RNA viruses, ssDNA viruses, shorter amplicons and plasmids. Our specialization and experience in virus sequencing allow us to customize sequencing projects for every client’s unique needs.
Sequencing data can be produced from various starting materials, including plasma, cell culture, RNA, and DNA. Virus starting material can be amplified using custom multiplex primer sets or unbiased amplification methods. In our MiSeq method, samples are molecularly barcoded and pooled together for a sequencing run. After images are processed and nucleotides are called on the MiSeq instrument, we bin the sequence reads by barcode tag. Sequenced sample data is typically sent back in the fastq file format but if a reference genome is available, we can also generate SAM/BAM (Sequence/Binary Alignment Map) files. Additional data analyses may be performed after consultation with Pathogen sequencing lead, Dr. Shelby O’Connor.
Viral pathogen stocks used to infect animals will be deep sequenced using Oxford Nanopore Technology long read sequencing free of charge* provided investigators agree to make this important information publicly available via the SRA database. BAM alignments will be generated based off a provided reference genome.
Please see our pathogen sequencing white paper (pdf) for additional details.
To learn more, contact Pathogen sequencing lead, Shelby O’Connor, PhD.
Illumina Pricing Examples:
| SERVICE | COST/SAMPLE** |
|---|---|
| SIV SEQUENCING FROM PLASMA – Qiagen | $213.63 |
| SIV SEQUENCING FROM PLASMA – Maxwell | $197.59 |
| VIRUS SEQUENCING MULTIPLEX TWO POOLS FROM PLASMA | $226.70 |
| VIRUS SEQUENCING FROM CDNA AMPLICONS GENERATED BY CLIENT | $237.16 |
| VIRUS INOCULUM | FREE |
**All pricing examples cover the cost of reagents, labor & BAM alignment. Prices shown are cost recovery rates for UW-Madison investigators. Other investigators are charged overhead rates ranging from 38.5% (federal or not-for-profit) to 72.5% (for-profit entities).
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