Mitochondrial Genomic Copy Number
The Core provides a mitochondrial copy number and nuclear genome copy number, as a surrogate for cell number, determination for a range of species by digital PCR.
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.
Absolute mtDNA quantitation dPCR workflow. Total genomic DNA is isolated from tissue/cells and mixed with proper assay components. The reactions are distributed across a chip with 20,000 856-pl wells. Template is diluted to a point where there is either 0 or 1 copies per well. Reactions are then cycled to end-point and fluorescence is read in each well. Those wells that were loaded with template are positive wells, while those wells that were filled without template are negative. Based on the count of fluorescent positive and negative wells and using a Poisson distribution, the number of target copies can be calculated per microliter. For a complete description of the dPCR methods see Masser et al., Age 2016 38:323-333.
Assays for human, mouse and rat genomes are available and can be generated for any species of interest.
Total sample DNA quantification is carried out using fluorescent-based PicoGreen assay (Life Technologies). Digital PCR is then performed according to manufacturer’s instructions by mixing 3.33 µl diluted template DNA (concentrations of DNA are first determined by performing test dilution series) with 16.5 µl Quantstudio 3D master mix, 3.33 µl TaqMan assay, and 9 µl water (32.16 µl [enough for two chips with excess]) (Life Technologies). Reactions are loaded onto Quantstudio 3D digital PCR chips using the Quantstudio 3D chip loader according to manufacturer’s instructions (Life Technologies). Chips are then sealed and cycled on a GeneAmp PCR system 9700 with a flatblock attachment using the following conditions; Stage 1, 96° C for 10 minutes, Stage 2, 60° C for 2 minutes then 98° C for 30 seconds, repeat Stage 2 39 times, Stage 3, 60° C for 2 minutes and an infinite 10° C hold. Chips are then read in the Quantstudio 3D chip reader to obtain raw fluorescent values (Life Technologies). Quality check of the chips and counting of positive and negative wells in order to determine copies/microliter are carried out on the Quantstudio 3D AnalysisSuite cloud software (https://apps.lifetechnologies.com/quantstudio3d/index.html; LifeTechnologies) using the Absolute Quantification module. Data can then be shared with other investigators through the cloud application or can be exported and sent in conventional spreadsheet format.
Mitochondrial genome copy number determination uses total DNA as input. No isolation of mitochondria is needed. Standard DNA isolation approaches appropriate to the tissue of interest (Trizol or spin columns) are fine. DNA should be quantified at least by spectrophotometry and ideally by Qubit. Concentrations should be >10ng/uL and at least 200ng of total DNA is required.
Mitochondrial genome copy number: $16/sample
Nuclear genome copy number: $16/sample
Mitochondrial Genomic Sequencing
The Core provides a mitochondrial genome sequencing for determination of mutations and deletions.
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, by long-range PCR, the mitochondrial genome 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.
The advantages of our approach are: 1) it does not require mitochondrial isolation, 2) it provides extremely high levels of sequencing depth (1,000-20,000X) using only a benchtop next-generation sequencer, 3) it requires very limited amount of sample (1ng DNA), and 4) the assay can be readily adapted to other model organisms of interest to the aging community. This approach allows a relatively inexpensive and high sample throughput analysis workflow for mtDNA sequencing with high levels of accuracy. Currently assays for human, mouse, and rat are available.
mtDNA sequencing workflow. A total of 1ng genomic DNA is subjected to long-range PCR of two overlapping mtDNA regions to selectively amplify mitochondrial genome. Long-range amplicons are subjected to transposome-mediated library construction, which simultaneously fragments and ligates sequencing adapters. Libraries are then amplified using primers with specific dual index sequences for multiplexed sequencing reactions. Libraries are then sequenced using paired-end 250 cycle benchtop sequencing). Low frequency variant detection is used to identify both protein coding and non-protein coding variants with frequencies > 0.5%
Quantitative accuracy of mt-DNA seq method.
In vitro generated synthetic mtDNA with mutations at four sites was mixed with unmutated DNA at percentages ranging from 0.1% to 100%. At all levels each of the four mutated sites was detected and accurately quantified.
For a full description of the methods see Masser et al., J Neurochem. 2017 143:595-608.
Example ideogram of mitochondrial genome sequencing data.
FASTQ files from the sequencing run are assessed for quality metrics with FastQC and then trimmed for quality with CLC Genomics Workbench. Trimmed reads are aligned and mapped with Bismark/Bowtie version adjusting alignment parameters for the highest sensitivity and against both strands. Data analysis and file management utilizes NGSUtils, samtools, and bedtools.
Data provided to core users include the FASTQ sequencing files and data files with location and level of each cytosine.
Mitochondrial genome sequencing uses total DNA as input. No isolation of mitochondria is needed. Standard DNA isolation approaches appropriate to the tissue of interest (Trizol or spin columns) are fine. DNA should be quantified at least by spectrophotometry and ideally by Qubit. Concentrations should be >10ng/uL and at least 200ng of total DNA is required.
Mitochondrial genome sequencing costs depend on the number of samples and the sequencing depth desired. Consult with the Core for a price estimate.
DNA modification services are designed such that either methylation or both methylation and hydroxymethylation can be examined across the entire genome, every gene, or select genes.
Click Image to Enlarge
Methods for base-specific cytosine modification quantitation. A Whole-genome bisulfite sequencing (WGBS) in general principle consists of bisulfite modification of genomic DNA and then creation of a sequencing library. Variants may switch the order of bisulfite conversion and library preparation. This approach gives quantitation across the genome but requires very large amounts of sequencing. B Bisulfite oligonucleotide capture sequencing (BOCS) is analogous to exome sequencing techniques. In this approach, a whole genome library is made and then bisulfite converted. Genomic regions of interest are then captured with oligonucleotide probes greatly enriching for regions of interest and thereby decreasing the amount of sequencing required. C Often, analysis of a specific genomic loci is desired, e.g., a gene of interest. With bisulfite amplicon sequencing (BSAS), the specific regions are amplified from bisulfite converted DNA and then made into a sequencing library. In effect, the PCR amplification greatly enriches for the region of interest.
For a full review and comparison of different approaches see Masser et al., Geroscience 2018 40:11-29.
Whole Genome Oxidative/Bisulfite Sequencing (WGox/BS)
For more directed hypothesie (e.g., does the promoter regions of a specific set of genes change with age) or to orthogonally confirm BOCS findings, we have developed BSAS (Masser et al., 2013) that allows one to measure the site specific levels of 5mC in specific regions (a few hundred bases to a few kilobases) of the genome.
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.
Genomic DNA is treated with bisulfite and/or oxidized and then bisulfite treated. Sequencing libraries are then made and whole genome sequencing is performed. For quantitative accuracy sequencing depths >10X are required. This approach can be applied to any species.
Whole genome bisulfite/oxidative bisulfite sequencing uses genomic DNA. Standard DNA isolation approaches appropriate to the tissue of interest (Trizol or spin columns) are fine. DNA should be quantified at least by spectrophotometry and ideally by Qubit. Concentrations should be >10ng/uL and at least 1ug of total DNA is required.
Whole genome analyzes typically cost between $2,500 and $7,500 per sample depending on the genome, number of samples, depth of sequencing coverage, and whether OxBS and BS are performed. Please consult with the core to develop a price estimate.
Oxidative/Bisulfite Oligonucleotide Capture Sequencing (ox/BOCS)
The BOCS assay gives investigators a genome-wide analysis of 5mC levels. This approach uses thousands of oligonucleotide probes designed to capture/target all gene promoters and all of the non-repeat region CpG islands, shores, and shelves of the genome sequencing analysis. We have probe sets for the human, mouse, and rat genomes and can design probes for any annotated genome. Currently, only the probe set for humans is commercially available. Our capture probe sets for rats are a resource unavailable elsewhere.
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.
Capture sets are available for human, mouse, and rat genomes. For a full description of BOCS see Masser et al., Age 2016 38:49.
Ox/BOCS sequencing uses genomic DNA. Standard DNA isolation approaches appropriate to the tissue of interest (Trizol or spin columns) are fine. DNA should be quantified at least by spectrophotometry and ideally by Qubit. Concentrations should be >10ng/uL and at least 1ug of total DNA is required.
BOCS analyzes typically cost between $1,500 and $3,500 per sample depending on the genome, number of samples, depth of sequencing coverage, and whether OxBS and BS are performed. Please consult with the core to develop a price estimate.
Oxidative/Bisulfite Amplicon Sequencing (ox/BSAS)
Bisulfite PCR primers are designed and tested against the regions(s) of interest. After conversion the region(s) of interest and amplified and then a sequencing library is made from the amplicons by tagmentation. Almost and region from any genome can be analyzed by this approach. For a full description of BSAS see Masser et al., Epigenetics & Chromatin. 2013 6:33.
Ox/BSAS sequencing uses genomic DNA. Standard DNA isolation approaches appropriate to the tissue of interest (Trizol or spin columns) are fine. DNA should be quantified at least by spectrophotometry and ideally by Qubit. Concentrations should be >10ng/uL and at least 1ug of total DNA is required.
BSAS analyzes typically cost between $150 and $750 per sample depending on the number of samples, the number of loci, depth of sequencing coverage, and whether OxBS and BS are performed. Please consult with the core to develop a price estimate.
Single Cell Transcriptomics
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.
While a great deal of attention has been focused on these droplet scRNA-Seq methods, a limitation is the depth of the transcriptome measured. Most 10X and ddSeq studies can measure around 1,000 mRNAs per cell - the most abundant genes. Some studies require a greater depth to the transcriptomic coverage. For these studies we will offer SMART-Seq (Takara) to generate cDNA, amplify, and create sequencing libraries. This approach uses template switching by reverse transcriptase to enrich for full-length cDNAs and add adapters of the first-strand cDNA. The approach is well suited for providing coverage across the transcript and not just from the 3’ end and enabling detection of splice variants and gene isoforms.
By their nature single cell transcriptomic studies are all custom designed studies that require optimization to maintain viable cells depending on the tissue or cell type. Consult with the Core on the design and optimization of cell isolation techniques and positive/negative control steps.
For local core users schedule an appointment with the Core to bring freshly isolated cells to the Core for library preparation. For our users that will require shipment of cells consult with the core on fixation approaches that prevent degradation of samples in shipment such as methanol fixation.
Single cell transcriptomic studies cost between $750 and $2,500 per sample depending on the process used, the number of samples, and sequencing depth. Please consult with the core to develop a price estimate.
Please leave your information below regarding the services you would like to discuss with the core directors. It is a requirement that those submitting a proposal for the Pilot Awards discuss their project with the appropriate core director before the proposal is submitted.
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© 2019 Oklahoma Nathan Shock Center on Aging