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Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next‐generation or massively parallel sequencing has made it possible to routinely obtain nucleotide‐level data with relative ease. These short‐read sequencing platforms offer deep coverage with unparalleled read accuracy in high‐complexity genomic regions but encounter numerous difficulties in the low‐complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy‐number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long‐read sequencing data on a pocket‐sized device known as the MinION. Nanopore‐based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor‐intensive, time‐consuming, and costly mainstay deep‐sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment‐free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR‐free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean‐up Support Protocol 3: Suggested data analysis pipeline

Background: The aging Mexican American (MA) population is the fastest growing ethnic minority group in the US. MAs have a unique metabolic-related risk for Alzheimer’s disease (AD) and mild cognitive impairment (MCI), compared to non-Hispanic whites (NHW). This risk for cognitive impairment (CI) is multifactorial involving genetics, environmental, and lifestyle factors. Changes in environment and lifestyle can alter patterns and even possibly reverse derangement of DNA methylation (a form of epigenetic regulation). Objective: We sought to identify ethnicity-specific DNA methylation profiles that may be associated with CI in MAs and NHWs. Methods: DNA obtained from peripheral blood of 551 participants from the Texas Alzheimer’s Research and Care Consortium was typed on the Illumina Infinium® MethylationEPIC chip array, which assesses over 850K CpG genomic sites. Within each ethnic group (N = 299 MAs, N = 252 NHWs), participants were stratified by cognitive status (control versus CI). Beta values, representing relative degree of methylation, were normalized using the Beta MIxture Quantile dilation method and assessed for differential methylation using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages in R. Results: Two differentially methylated sites were significant: cg13135255 (MAs) and cg27002303 (NHWs) based on an FDR p < 0.05. Three suggestive sites obtained were cg01887506 (MAs) and cg10607142 and cg13529380 (NHWs). Most methylation sites were hypermethylated in CI compared to controls, except cg13529380 which was hypomethylated. Conclusion: The strongest association with CI was at cg13135255 (FDR-adjusted p = 0.029 in MAs), within the CREBBP gene. Moving forward, identifying additional ethnicity-specific methylation sites may be useful to discern CI risk in MAs.

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available — the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, long-read data. To demonstrate the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs with >90% accuracy. These results demonstrate that nanopore sequencing platforms are capable of revealing additional variation in and around STR loci depending on read coverage. As the first long-read platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

Background Alzheimer’s disease (AD) and type 2 diabetes (T2D) are among the leading causes of mortality among the growing aging Mexican American population (≥ 65 years old) in the US (Vega et al. 2017). In comparison to their non‐Hispanic white counterparts who most likely develop inflammation associated AD, aging Mexican Americans have an earlier onset of AD and metabolism related predisposition for AD (O'Bryant et al. 2010). Mild cognitive impairment (MCI) is a phenotype that often leads to AD and is also prevalent in this cohort (O'Bryant et al. 2013). The risks for AD, MCI and T2D are multifactorial, involving a form of epigenetic regulation called methylation where a methyl group is added to the cytosine base in DNA (Shao et al. 2017; Juvinao‐Quintero et al. 2019). We aim to elucidate an epigenetic association between cognitive impairment (identified here as AD and MCI), and T2D that is unique to the Mexican American population. Method A total of 551 aging participants from the Texas Alzheimer’s Research and Care Consortium (TARCC) consisting of 252 non‐Hispanic white individuals and 299 Mexican Americans were selected, after quality control. First, participants were stratified into groups of individuals diagnosed with cognitive impairment (CI) alone and controls without CI within each ethnic group. Secondly, this cohort was stratified into individuals with T2D alone and controls without T2D. Thirdly, participants were stratified into those with both CI and T2D versus normal healthy controls. Lastly, any differential methylation associated with each ethnic group will be compared and contrasted. Peripheral blood drawn from participants was used to obtain individual methylation profiles using the Illumina Infinium MethylationEPIC chip array. Differential methylation was assessed using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages in R. The Beta MIxture Quantile dilation (BMIQ) method was used for data normalization. Result Results will be analyzed using gene set enrichment and pathway analysis tools. Conclusion Identifying possible methylation sites associated with CI and T2D could contribute towards developing ethnicity‐specific biomarkers for Mexican Americans.

Background Alzheimer’s disease (AD) is a complex neurodegenerative disorder that disproportionately burdens Mexican Americans (MAs) due to a combination of population‐specific environmental exposures and genetic risk factors. In addition to AD, MAs have a higher prevalence of metabolic comorbidities such as type 2 diabetes (T2D) that have been implicated in accelerated age‐related cognitive impairment. Tissue‐specific mitochondrial dysfunction and oxidative stress are early and prominent features of both AD and T2D that propagate systemic inflammation as these diseases progress. Despite the shared pathophysiology, we do not fully understand the underlying molecular mechanisms linking cognitive decline and metabolic stress, especially in understudied minority populations. Mitochondrial dysfunction may serve as the biological basis of the AD health disparity in MAs who experience earlier onset of symptoms and have an overrepresentation of T2D compared to non‐Hispanic whites (NHWs). This project aims to study T2D‐related risk for cognitive impairment by assessing the packages that neurons secrete to communicate with cells in the periphery. These neuronal‐enriched exosomes (NEEs) can cross the blood‐brain barrier and are thus capable of mediating systemic inflammation in response to CNS‐sourced stress through their mitochondrial DNA (mtDNA) cargo. We hypothesize that plasma NEE mtDNA load will reflect cognitive status and T2D comorbidity over time. Methods Exosomes of neuronal origin were enriched via CD171‐based immunocapture in longitudinal plasma samples (two timepoints, 5 years apart) from 348 Texas Alzheimer’s Research and Care Consortium (TARCC) participants stratified by race/ethnicity (MA/NHW), cognitive status (AD/MCI/NC), and T2D comorbidity (+/‐). The success of NEE isolation was assessed using nanoparticle tracking analysis (NTA) and immunoblotting against CD171 and CD81 (canonical exosome surface marker). Following extraction of total DNA from NEEs, absolute mtDNA load and deletion ratio were estimated using a real‐time qPCR assay targeting sites within the minor (D‐loop) and major (ND4) arcs. Results Herein, we demonstrate the ability to isolate NEEs from plasma and quantify their mtDNA cargo. Resultant values were analyzed for longitudinal differences and correlation with disease status based on various neuropsychological measures. Conclusion As the first effort to assess mtDNA in NEEs, this project provides novel insight into how T2D confers risk for AD across different populations over time.