Molecular Phenotyping & Genotyping Core

The Core extracts DNA from human blood samples collected by Joslin investigators as part of their IRB-approved projects. An automated DNA extraction protocol uses the Qiacube Connect (Qiagen), a robotic workstation that reduces manual processing time from three to one hour, lowers input requirements to 200 µl of whole blood or packed cells, and maintains high DNA quality and yield, with an average output of 7µg of DNA from 200 µl of buffy coat. Investigators can also be trained for self-use for RNA and DNA extractions. A Tissue Lyser (Qiagen) is available for homogenizing tissues and a Covaris ultrasonicator is available for DNA fragmentation.

The Core maintains a Nanodrop and Nanodrop 8 spectrophotometers for routine analysis of DNA and RNA content and quality (open usage, not tracked). For more detailed analysis, the Core hosts an Agilent 4150 TapeStation which allows fully automated processing of 16 samples at a time.

For analysis of expression and/or allelic discrimination protocols, the Core provides and maintains equipment for real-time quantitative PCR (Thermo Fisher QuantStudio 6 and two QuantStudio 6 Pro instruments) analyses. To quantify absolute 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 and maintains a Thermo Fisher QuantStudio Absolute Q PCR Instrument. Core personnel provide training and assistance with troubleshooting for these self-use instruments.

The Core supports single-cell/single-nucleus RNA sequencing using the 10X Chromium platform. Interested users will receive training from Core personnel, 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.

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).

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. Gene Panel for Next-Gen Sequencing of Genetic Variants

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.

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.