Immunology, Retrovirology and Cure Core

UW/Fred Hutch CFAR Immunology Core has used CFAR Administrative Supplement funding to purchase a 6-color Naica droplet digital PCR (dPCR) system. The system provides absolute counts of target copies using up to 6 fluorescent dyes and no standard curve is required.

Workflow: Primers, probes and template are combined with PCR master-mix and transferred to a chip. Partitioning and thermal-cycling, take place in the Geode instrument and the chip is transferred to the Prism instrument for scanning and imaging. Raw data can be viewed and analyzed in the provided Crystal Miner analysis software.

Applications: ddPCR is the ideal technology to quantify nucleic acids with high precision; applications include rare event detection, whole genome amplification and copy number variation. In the Hladik lab, this instrument will allow the team to accurately measure intact HIV proviral reservoirs, get the most HIV-related information from each sample by highly multiplexing, and recover samples after analysis for subsequent sequencing. Multi-color-detection also allows combination of reference assays with assays of interest in the same ddPCR reaction (increasing normalization accuracy), testing of multiple mutational variants in the same reaction, and conservation of limited sample material (e.g., blood from infants or mucosal specimens).

Use: The equipment is now available for use in the Center for Reproductive Sciences in the UW SLU Campus (850 Republican St), building S, room 220. The accompanying analysis software (Crystal Miner) can be installed on a separate PC for post-run analysis outside the lab. The Hladik group has extensive experience running routine ddPCR assays, using ddPCR equipment creatively to design and optimize specialized assays, as well as analyze and visualize raw results using R or excel.

Interested CFAR users will either contract with us to design and run assays for them (clinical scientists), or we will train them to use the equipment, with ongoing technical and assay design assistance (laboratory scientists).

interested parties should contact Florian Hladik at fhladik@fredhutch.org

We provide researchers consultation access to Core scientists with leading edge expertise in all current nucleic acid sequencing methodologies, including Sanger, Illumina, PacBio, and Oxford Nanopore platforms. We also provide legacy support for the 454/pyrosequencing platform. Consulting on HIV genome sequence analysis includes bioinformatic pipeline development, high-performance computing power, and interpretive support from Core staff.

Our scientists offer consulting to investigators on the experimental design, optimal specimen acquisition, collection and processing, experimental aspects of clinical trial development, data processing and data interpretation, focusing on experiments that acquire molecular sequence data. Areas of expertise include virologic assays, nucleic acid sequence analysis (see below), phylogenetics, molecular epidemiology, viral integration site analysis, and more.