Molecular Phenotyping & Genotyping Core

The Core extracts DNA from blood or saliva samples collected by Joslin investigators using a mini-column based protocol (QIAamp Blood Midi Kit).

The Core provides a single nucleotide polymorphism (SNP) typing service for projects including up to 64 SNPs. Typically, these are SNPs that have been identified by genome-wide association studies (GWAS) or resequencing projects as being associated with diabetes or its complications, and for which replication is being sought in other studies. The service is based on individual TaqMan assays, which are run by means of a robotic liquid handling workstation (Tecan EVO 150).

Extraction of high-quality DNA from human specimens, including blood samples.

This genetic assay simultaneously screens the coding sequences of all known genes for monogenic diabetes (MODY, neonatal diabetes, etc.) for mutations, and at the same time genotype all known loci for type 1 diabetes (T1D, excluding HLA), type 2 diabetes (T2D) and related quantitative metabolic traits, and diabetic complications. The current version (v3.1) includes 30 genes for monogenic diabetes (Table 1) along with 762 SNPs (91 for T1D, 133 for T2D and related metabolic traits, 221 for coronary heart disease, 41 for diabetic nephropathy, 14 for diabetic retinopathy, and 262 ancestry informative SNPs allowing adjustment for population stratification).Core personnel prepare genomic DNA libraries and use RNA probes to enrich target genomic regions for sequencing by PCR. The Core then facilitates Next-gen-sequencing of enriched libraries through one of Harvard’s several cores.

Table 1. Genetic Assay for Clinical Studies

The core maintains a Nanodrop spectrophotometer for routine analysis of DNA and RNA content and quality (open usage, not tracked). For more detailed analysis, the core hosts and maintains an Agilent 2100 Bioanalyzer system for sizing, quantitation, and quality control of DNA and RNA samples and libraries.

The Core supports a Bruker MALDI Biotyper Microflex™LT/SH CM System, a bench-top MALDI-TOF mass spectrometer designed to rapidly and accurately identify bacteria and fungi from pure culture for microbiome research. Downstream steps typically include the generation of strain/isolate catalogs from a given microbiome, or the targeted isolation of key microbes so they can be characterized or genetically manipulated individually.

A TissueLyser (Qiagen) is maintained for investigator use. This instrument is designed for reproducible disruption of multiple tissue samples or biopsy through high-speed shaking in plastic tubes with a range of stainless steel, tungsten carbide, or glass beads for subsequent downstream molecular analysis.

For analysis of expression and/or allelic discrimination protocols, the Core provides and maintains equipment for real-time quantitative PCR analyses and provides training and assistance with troubleshooting.

The core provides a Bio-Rad Digital PCR platform for Digital detection of RNA, DNA, and noncoding RNA species to enable investigators to analyze gene expression in small samples without potential biases introduced by amplification and without the need for relative quantification using a housekeeping gene.

The Core provides analysis of metabolic flux using the Seahorse XF24 and XF96 instruments, permitting metabolic phenotyping of cultured cells, human/mouse iPS cells, tissue explants, isolated mitochondria, and whole blood and other primary samples from clinical research participants.

The core supports single-cell/single-nucleus RNA sequencing using the 10X Chromium platform. Interested users will receive training from core personnel (Dr. Qiong Zhou), including sharing and review of validated tissue-specific protocols for sample preparation to generate single-cell or single-nucleus suspensions (for frozen tissue). The core provides a ready-to-sequence library for sequencing at an Illumina-compatible sequencing core of the user’s choice. Once RNA-seq data are available, data can be transferred to the Joslin Bioinformatics Core, which is highly experienced in single-cell RNA-seq data processing. The core also supports single-cell/single-nucleus ATAC sequencing for analysis of chromatin structure; this can be performed independently or simultaneously with RNA-sequencing using the single-cell Multiome kits.

A major component of core services is the education of investigators, fellows, students, and technicians regarding experimental design, optimization of sample preparation, and data analysis. Core directors and staff work meet with investigators to provide assistance with experimental design, instrument use, and data interpretation.