University of Wisconsin–Madison

Genetics Services Unit

The WNPRC Genetic Services team includes, from the left, Julie Karl, Hailey Bussan, Roger Wiseman, Shelby O'Connor, Dave O'Connor, Kristi Hall, Dane Gellerup, Michael Graham. (J. Lenon image)

The WNPRC Genetics Services Unit offers two main services: nonhuman primate MHC genotyping and pathogen sequencing. High-throughput MHC genotyping assays for Mauritian cynomolgus macaques, Indian rhesus macaques, and other nonhuman primates are available, as well as full-length MHC transcript sequencingPathogen sequencing is also available, with an emphasis on simian immunodeficiency viruses like SIVmac239, SIVmac251, and SIVsmE660.

CONTACT INFORMATION

Services Supported

MHC genotyping

Since 2009, Genetics Services has provided high-throughput MHC genotyping for investigators utilizing nonhuman primates to study cellular immunity and transplantation tolerance. Our sequence-based approach was originally described in a Nature Medicine manuscript using Roche/454 Life Sciences sequencing; it was later adapted to use the Illumina MiSeq sequencing platform for improved throughput. The MHC region of macaques has ‘unparalleled complexity’ versus the human MHC, with each animal expressing a variable number of MHC class I alleles. There are also thousands of macaque MHC allelic variants described to date. We assist researchers in understanding this complex diversity by sequencing either the most polymorphic segments of MHC genes or the full-length transcripts, then grouping resulting sequences into simplified haplotypes to indicate differing complements of highly transcribed alleles. These haplotype designations can be used to more easily identify pairs or groups of animals that share multiple MHC alleles. Standard genotyping reports also include the full list of detected alleles, in addition to the haplotype designations.

Our standard MiSeq assays all focus on the most polymorphic segments of MHC genes, providing lineage-level allelic resolution. This level of resolution is typically sufficient for balancing study and control groups and for determining degree of MHC mismatch for transplant studies. For instances where full resolution of the specific MHC allelic variant is important, we also provide PacBio SMRT sequencing of full-length MHC transcripts. The Genetics Services staff can assist in determining which assay would be most beneficial for a particular study.

Mauritian-origin cynomolgus macaque Fluidigm/MiSeq MHC class I and class II (DRB, DQA/B, DPA/B) genotyping

The MHC complement of the entire population of Mauritian cynomolgus macaques is limited to seven haplotypes, referred to as M1-M7. This restricted MHC diversity greatly simplifies genotyping analysis. We have developed a MiSeq sequence-based genotyping assay for Mauritian cynomolgus macaques using a Fluidigm Access Array microfluidics chip to simultaneously PCR amplify exon 2 of MHC class I, class II DRB, class II DQA/B, and class II DPA/B loci from genomic DNA. Sample-specific barcodes are added to each sample, and amplicons for approximately 200 samples are pooled together for MiSeq sequencing. After images are processed and nucleotides are called on the MiSeq instrument, we bin the sequence reads by barcode tag and perform a comparison of the sequence reads against a curated in-house database of Mauritian cynomolgus macaque MHC sequences. These results are passed through an automated pipeline for calling haplotypes based on the detection of diagnostic alleles for the M1-M7 haplotypes for each region (A, B, DRB, DQA/B, DPA/B). See our MHC genotyping white paper for additional details.

Indian-origin rhesus macaque Fluidigm/MiSeq MHC class I and class II (DRB, DQA/B, DPA/B) genotyping

Indian rhesus macaques from multiple breeding centers throughout the country also exhibit somewhat restricted MHC diversity. A very limited number of A, B, and DRB haplotypes account for over half of the MHC diversity of Indian rhesus macaques available for research protocols. We can apply the MiSeq sequence-based genotyping assay to Indian rhesus macaques using a Fluidigm Access Array microfluidics chip to simultaneously PCR amplify exon 2 of MHC class I, class II DRB, class II DQA/B, and class II DPA/B loci from genomic DNA. Sample-specific barcodes are added to each sample, and amplicons for approximately 200 samples are pooled together for MiSeq sequencing. After images are processed and nucleotides are called on the MiSeq instrument, we bin the sequence reads by barcode tag and perform a comparison of the sequence reads against a curated in-house database of rhesus macaque MHC sequences. Haplotypes are then manually determined for each region (A, B, DRB, DQA/B, DPA/B). See our MHC genotyping white paper for additional details.

Other nonhuman primate MiSeq MHC class I and class II DRB genotyping

We have performed MiSeq MHC class I and class II DRB genotyping on many other populations of nonhuman primates to date, such as Chinese rhesus macaques, Indonesian cynomolgus macaques, cynomolgus macaques from Chinese breeding facilities, and pig-tailed macaques. We PCR amplify exon 2 of MHC class I and class II DRB (not class II DQA/B or DPA/B) from cDNA synthesized from total RNA to remove untranslated pseudogenes from the analysis. Sample-specific barcodes are added to each sample, and amplicons for approximately 200 samples are pooled together for MiSeq sequencing. After images are processed and nucleotides are called on the MiSeq instrument, we bin the sequence reads by barcode tag and perform a comparison of the sequence reads against a curated in-house database of species-specific MHC sequences, then generate haplotypes wherever possible. Because these populations are overall more diverse than Indian rhesus and Mauritian cynomolgus macaques, there is an increased chance we will encounter novel haplotypes during routine MiSeq genotyping. In populations with particularly small databases of known MHC alleles, the MiSeq assay may be only minimally informative since results are scored through comparison to the allele database. Alternatively, the PacBio full-length assay can be used to unambiguously resolve novel alleles in these populations. See our MHC genotyping white paper for additional details.

PacBio full-length transcript MHC genotyping and allele discovery

For diverse nonhuman primate populations with currently small databases of known MHC alleles, like Chinese rhesus macaques, pig-tailed macaques, and cynomolgus macaques of non-Mauritian origin, we perform full-length transcript sequencing using PacBio single molecule real-time (SMRT) sequencing. Since the entire coding region is analyzed, the specific, unambiguous allelic variant is determined via this assay. We PCR amplify full-length transcripts for MHC class I (A and B) and/or MHC class II (DRA/B, DQA/B, DPA/B) from cDNA templates generated from total RNA. PacBio barcode identifier sequence tags are incorporated via the PCR primers, and multiple samples are pooled together for PacBio SMRT sequencing. After sequence reads are processed on the PacBio instrument, we bin the reads by barcode tag and perform a comparison of the sequence reads against a curated in-house database of species-specific MHC sequences. Any reads not matching a database sequence are further processed to create clusters of identical reads, and the cluster sequences are then manually curated to determine if they are extensions of previously identified alleles or putative novel alleles. Results are reported as a list of full-resolution MHC alleles, and haplotypes are assigned wherever possible. See our MHC genotyping white paper for additional details.

Viral RNA genome characterization with Illumina MiSeq technology

We specialize in RNA viral genome sequencing with an emphasis on simian immunodeficiency viruses. We use Illumina MiSeq technology to sequence the entire coding sequence of RNA viruses, shorter amplicons, and plasmids. This data can be produced from various starting materials, including plasma, cell culture, RNA, and DNA. Each genome is molecularly barcoded, and 20-30 viral genomes are pooled together for each MiSeq run. After images are processed and nucleotides are called on the MiSeq instrument, we bin the sequence reads by barcode tag. In all cases, we can provide fastq files for each read. If a reference genome is available, we can also generate BAM or SAM alignments. Additional data analyses may be performed after discussions with Genetics Services. See our pathogen sequencing white paper for additional details.

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