Genomic Sciences Core
The Genomic Sciences Core provides researchers with state-of-the-art novel technologies developed to address fundamental questions of how genomes change with age, focusing on DNA modification patterns and the accumulation of mutations and deletions in the mitochondrial genome (mtDNA). The core also provides Geroscience researchers with unique assays of DNA modifications and mtDNA heteroplasmy typically not present in individual laboratories or institutional core facilities.
Mitochondrial Genomic Copy Number
Changes in mitochondrial genome number have been observed with aging but traditional techniques have been limited as they only provide a relative quantitation. Using digital PCR to ‘count’ mitochondrial genomes provides an absolute quantitation, allowing data to be compared study to study. Nuclear genome counting in parallel as a surrogate for cell number allows for normalization to cell number.
Mitochondrial Genomic Sequencing
Heteroplasmic mutations in the mitochondrial genomes accumulate with age. Modern sequencing techniques allow quantitation and localization of mutations and deletions. The approach used for mitochondrial sequencing is to amplify the mitochondrial genome by long-range PCR to enrich for the mtDNA and focus sequencing. By sequencing the mitochondrial genome at high depth (~1,000X coverage), mutations occurring at rate as low as 0.1 to 1% can be quantified. We have validated this method with in vitro derived standards and can accurately identify and quantitate specific large-scale deletions and single nucleotide variants.
Whole Genome Oxidative/Bisulfite Sequencing (WGox/BS)
The most comprehensive analysis of the DNA modifications provides base-specific quantitation of DNA methylation in both CG and CH contexts. This can be paired with oxidative bisulfite sequencing to measure hydroxymethylation.
Oxidative/Bisulfite Oligonucleotide Capture Sequencing (Ox/BOCS)
Given the extensive amount of sequencing required for whole genome analyses we have optimized a capture based approach to focus sequencing on relevant gene promoters and enhancers. Much like exome sequencing this decreases the amount of sequencing required but still provides data from across the genome (1-2million CG sites). This can be paired with oxidiative conversion to measure hydroxymethylation.
Oxidative/Bisulfite Amplicon Sequencing (Ox/BSAS)
For many studies a specific gene or loci is of interest. By amplifying the region(s) of interest sequencing can be performed on large numbers of samples to a great depth using only a benchtop sequencer. This can also be paired with oxidative conversion to measure hydroxymethylation.
The design of the services for Single-Cell RNA Sequencing (scRNA-Seq) is in three tiers so that investigators can tailor their studies to the number of cells that need to be analyzed, the depth of transcriptome coverage, and their available resources. At the broadest reach, 10X Genomics Chromium instrumentation services enable analysis of 1,000s of cells per sample with 100s to low 1,000s of mRNAs per cell. However, these are significant studies and cell preparation approaches must be optimized for each project. A less expensive, lower cell number (100s) approach such as Illumina/BioRad ddSeq is an attractive alternative for initial work.
Willard "Bill" Freeman, Ph.D.
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