Nanopore Sequencing Reads Research Articles

NanoCore: core-genome-based bacterial genomic surveillance and outbreak detection in healthcare facilities from Nanopore and Illumina data.

Genomic surveillance involves sequencing the genomes and measuring the relatedness of bacteria from different patients or locations in the same healthcare facility, enabling an improved understanding of pathogen transmission pathways and the detection of "silent" outbreaks that would otherwise go undetected. It has become an indispensable tool for the detection and prevention of healthcare-associated infections and is routinely applied by many healthcare institutions. The earlier an outbreak or transmission chain is detected, the better; in this context, the Oxford Nanopore sequencing technology has important potential advantages over traditionally used short-read sequencing technologies, because it supports "real-time" data generation and the cost-effective "on demand" sequencing of small numbers of bacterial isolates. The analysis of Nanopore sequencing data, however, can be challenging. We present NanoCore, a user-friendly software for genomic surveillance that works directly based on Nanopore sequencing reads in FASTQ format, and demonstrate that its accuracy is equivalent to traditional gold standard short read-based analyses.

Characterization of the complete mitogenome of Tiarella polyphylla, commonly known as Asian foamflower: insights into the multi-chromosomes structure and DNA transfers

BackgroundTiarella polyphylla D. Don has been traditionally used to cure asthma and skin eruptions. However, the sequence and the structure of the mitogenome of T. polyphylla remained elusive, limiting the genomic and evolution analysis based on the mitogenome.ResultsUsing a combination of Illumina and Nanopore sequencing reads, we de novo assembled the complete mitogenome of T. polyphylla. In addition to unveiling the major configuration of the T. polyphylla mitogenome was three circular chromosomes with lengths of 430,435 bp, 126,943 bp, and 55,269 bp, we revealed five (R01-R05) and one (R06) repetitive sequence could mediate the intra- and inter-chromosomal recombination, respectively. Furthermore, we identified 208 short and 25 long tandem segments, seven cp-derived mtDNAs, 106 segments of mtDNAs transferred to the nuclear genome, and 653 predicted RNA editing sites. Based on the sequence of the mitogenomes, we obtained the resolved phylogeny of the seven Saxifragales species.ConclusionsThese results presented the mitogenome features and expanded its potential applications in phylogenetics, species identification, and cytoplasmic male sterility (CMS) in the future.

Quantitative analysis of dose dependent DNA fragmentation in dry pBR322 plasmid using long read sequencing and Monte Carlo simulations

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose–effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.

Nanopore duplex sequencing as an alternative to Illumina MiSeq sequencing for eDNA-based biomonitoring of coastal aquaculture impacts

Oxford Nanopore Technologies has recently launched a duplex sequencing strategy and announced an improved error rate which is in a similar order of magnitude as Illumina sequencing. We therefore conducted a pilot study to assess whether Nanopore duplex sequencing has potential to be used as a technology in eDNA-based marine biomonitoring. Specifically, we investigated bacterial communities of sediment samples collected from Atlantic salmon (Salmo salar) aquaculture installations and compared the ecological trends obtained from short Illumina (V3-V4 region of the 16S rRNA gene) and long Nanopore (full length 16S rRNA gene) sequence reads. The obtained duplex rate of Nanopore amplicon reads with a Phred score ≥ 30 was 36%, notably higher compared to previous reports from bacterial genome sequencing. When inferring alpha- and beta-diversity from Illumina ASVs and Nanopore OTUs, we found highly congruent ecological patterns. Only when collating ASVs and OTUs across taxonomic ranks, beta-diversity analyses of Illumina-data slightly changed, due to the difficulties to assign a taxonomy to short sequence reads. While on family rank, both sequence datasets had good agreement, genus-assignments of Illumina data were critical, resulting in higher disagreement between the two protocols. Our data provide evidence that eDNA-based monitoring of aquaculture-related environmental impacts could equally well be conducted with the improved Nanopore duplex sequencing. We discuss to what extent eDNA-based biomonitoring could benefit from long-read information.

Automated detection and classification of polioviruses from nanopore sequencing reads using piranha.

Widespread surveillance, rapid detection, and appropriate intervention will be critical for successful eradication of poliovirus. Using deployable next-generation sequencing (NGS) approaches, such as Oxford Nanopore Technologies' MinION, the time from sample to result can be significantly reduced compared to cell culture and Sanger sequencing. We developed piranha (poliovirus investigation resource automating nanopore haplotype analysis), a 'sequencing reads-to-report' solution to aid routine poliovirus testing of both stool and environmental samples and alleviate the bioinformatic bottleneck that often exists for laboratories adopting novel NGS approaches. Piranha can be used for efficient intratypic differentiation of poliovirus serotypes, for classification of Sabin-like polioviruses, and for detection of wild-type and vaccine-derived polioviruses. It produces interactive, distributable reports, as well as summary comma-separated values files and consensus poliovirus FASTA sequences. Piranha optionally provides phylogenetic analysis, with the ability to incorporate a local database, processing from raw sequencing reads to an interactive, annotated phylogeny in a single step. The reports describe each nanopore sequencing run with interpretable plots, enabling researchers to easily detect the presence of poliovirus in samples and quickly disseminate their results. Poliovirus eradication efforts are hindered by the lack of real-time detection and reporting, and piranha can be used to complement direct detection sequencing approaches.

Evaluation of Long-Read Sequencing Simulators to Assess Real-World Applications for Food Safety.

Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes are routinely responsible for severe foodborne illnesses in the United States. Current identification methods utilized by the U.S. Food Safety Inspection Service require at least four days to identify STEC and six days for L. monocytogenes. Adoption of long-read, whole genome sequencing for food safety testing could significantly reduce the time needed for identification, but method development costs are high. Therefore, the goal of this project was to use NanoSim-H software to simulate Oxford Nanopore sequencing reads to assess the feasibility of sequencing-based foodborne pathogen detection and guide experimental design. Sequencing reads were simulated for STEC, L. monocytogenes, and a 1:1 combination of STEC and Bos taurus genomes using NanoSim-H. At least 2500 simulated reads were needed to identify the seven genes of interest targeted in STEC, and at least 500 reads were needed to detect the gene targeted in L. monocytogenes. Genome coverage of 30x was estimated at 21,521, and 11,802 reads for STEC and L. monocytogenes, respectively. Approximately 5-6% of reads simulated from both bacteria did not align with their respective reference genomes due to the introduction of errors. For the STEC and B. taurus 1:1 genome mixture, all genes of interest were detected with 1,000,000 reads, but less than 1x coverage was obtained. The results suggested sample enrichment would be necessary to detect foodborne pathogens with long-read sequencing, but this would still decrease the time needed from current methods. Additionally, simulation data will be useful for reducing the time and expense associated with laboratory experimentation.

Genomic Sequence Resource of Talaromyces albobiverticillius, the Causative Pathogen of Pomegranate Pulp Rot Disease.

Talaromyces albobiverticillius, a prominent pathogen responsible for pomegranate pulp rot disease, inflicts significant damage on Punica granatum L. Besides its pathogenicity, this fungus possesses the potential to produce substantial amounts of red pigments, making it promising for industrial applications. This study presents the genome annotation of T. albobiverticillius field strain Tp-2, isolated from pomegranates. The genome assembly, generated through a combination of Oxford Nanopore and Illumina sequencing reads, yielded a high-quality assembly with 14 contigs, featuring an N50 length of 4,594,200 bp. The complete genome of strain Tp-2 spans 38,354,882 bp, with a GC content of 45.78%. Importantly, the assembly exhibits remarkable integrity, with 98.3% of complete Benchmarking Universal Single-Copy Orthologs validating genome completeness. Genome prediction analysis reveals the presence of 10,380 protein-coding genes. To our knowledge, this study is the first report on the genome sequence of T. albobiverticillius, offering valuable insights into its genetic variation and molecular mechanisms of pigment production.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former’s inefficiency and the latter’s inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional ‘Correction’ mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in ‘Correction’ mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Genome Report: chromosome-scale genome assembly of the African spiny mouse (Acomys cahirinus).

There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Balancing read length and sequencing depth: Optimizing Nanopore long-read sequencing for monocots with an emphasis on the Liliales.

We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results. Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.

Telomere-to-telomere assembly of diploid chromosomes with Verkko.

The Telomere-to-Telomere consortium recently assembled the first truly complete sequence of a human genome. To resolve the most complex repeats, this project relied on manual integration of ultra-long Oxford Nanopore sequencing reads with a high-resolution assembly graph built from long, accurate PacBio high-fidelity reads. We have improved and automated this strategy in Verkko, an iterative, graph-based pipeline for assembling complete, diploid genomes. Verkko begins with a multiplex de Bruijn graph built from long, accurate reads and progressively simplifies this graph by integrating ultra-long reads and haplotype-specific markers. The result is a phased, diploid assembly of both haplotypes, with many chromosomes automatically assembled from telomere to telomere. Running Verkko on the HG002 human genome resulted in 20 of 46 diploid chromosomes assembled without gaps at 99.9997% accuracy. The complete assembly of diploid genomes is a critical step towards the construction of comprehensive pangenome databases and chromosome-scale comparative genomics.

Reference-free lossless compression of nanopore sequencing reads using an approximate assembly approach

The amount of data produced by genome sequencing experiments has been growing rapidly over the past several years, making compression important for efficient storage, transfer and analysis of the data. In recent years, nanopore sequencing technologies have seen increasing adoption since they are portable, real-time and provide long reads. However, there has been limited progress on compression of nanopore sequencing reads obtained in FASTQ files since most existing tools are either general-purpose or specialized for short read data. We present NanoSpring, a reference-free compressor for nanopore sequencing reads, relying on an approximate assembly approach. We evaluate NanoSpring on a variety of datasets including bacterial, metagenomic, plant, animal, and human whole genome data. For recently basecalled high quality nanopore datasets, NanoSpring, which focuses only on the base sequences in the FASTQ file, uses just 0.35–0.65 bits per base which is 3–6times lower than general purpose compressors like gzip. NanoSpring is competitive in compression ratio and compression resource usage with the state-of-the-art tool CoLoRd while being significantly faster at decompression when using multiple threads (> 4times faster decompression with 20 threads). NanoSpring is available on GitHub at https://github.com/qm2/NanoSpring.

Mobile and Self-Sustained Data Storage in an Extremophile Genomic DNA.

DNA has been pursued as a novel biomaterial for digital data storage. While large-scale data storage and random access have been achieved in DNA oligonucleotide pools, repeated data accessing requires constant data replenishment, and these implementations are confined in professional facilities. Here, a mobile data storage system in the genome of the extremophile Halomonas bluephagenesis, which enables dual-mode storage, dynamic data maintenance, rapid readout, and robust recovery. The system relies on two key components: A versatile genetic toolbox for the integration of 10-100kb scale synthetic DNA into H. bluephagenesis genome and an efficient error correction coding scheme targeting noisy nanopore sequencing reads. The storage and repeated retrieval of 5KB data under non-laboratory conditions are demonstrated. The work highlights the potential of DNA data storage in domestic and field scenarios, and expands its application domain from archival data to frequently accessed data.

Allele detection using k-mer-based sequencing error profiles.

For genotype and haplotype inference, typically, sequencing reads aligned to a reference genome are used. The alignments identify the genomic origin of the reads and help to infer the absence or presence of sequence variants in the genome. Since long sequencing reads often come with high rates of systematic sequencing errors, single nucleotides in the reads are not always correctly aligned to the reference genome, which can thus lead to wrong conclusions about the allele carried by a sequencing read at the variant site. Thus, allele detection is not a trivial task, especially for single-nucleotide polymorphisms and indels. To learn the characteristics of sequencing errors, we introduce a method to create an error model in non-variant regions of the genome. This information is later used to distinguish sequencing errors from alternative alleles in variant regions. We show that our method, k-merald, improves allele detection accuracy leading to better genotyping performance as compared to the existing WhatsHap implementation using edit-distance-basedallele detection, with a decrease of 18% and 24% in error ratefor high-coverage Oxford Nanopore and PacBio CLR sequencing reads for sample HG002, respectively. We additionally observed a prominent improvement in genotyping performance for sequencing data with low coverage. For 3 coverage Oxford Nanopore sequencing data, the genotyping error rate reduced from 34% to 31%, corresponding to a 9% decrease. https://github.com/whatshap/whatshap.

NanoSNP: a progressive and haplotype-aware SNP caller on low-coverage nanopore sequencing data.

Oxford Nanopore sequencing has great potential and advantages in population-scale studies. Due to the cost of sequencing, the depth of whole-genome sequencing for per individual sample must be small. However, the existing single nucleotide polymorphism (SNP) callers are aimed at high-coverage Nanopore sequencing reads. Detecting the SNP variants on low-coverage Nanopore sequencing data is still a challenging problem. We developed a novel deep learning-based SNP calling method, NanoSNP, to identify the SNP sites (excluding short indels) based on low-coverage Nanopore sequencing reads. In this method, we design a multi-step, multi-scale and haplotype-aware SNP detection pipeline. First, the pileup model in NanoSNP utilizes the naive pileup feature to predict a subset of SNP sites with a Bi-long short-term memory (LSTM) network. These SNP sites are phased and used to divide the low-coverage Nanopore reads into different haplotypes. Finally, the long-range haplotype feature and short-range pileup feature are extracted from each haplotype. The haplotype model combines two features and predicts the genotype for the candidate site using a Bi-LSTM network. To evaluate the performance of NanoSNP, we compared NanoSNP with Clair, Clair3, Pepper-DeepVariant and NanoCaller on the low-coverage (∼16×) Nanopore sequencing reads. We also performed cross-genome testing on six human genomes HG002-HG007, respectively. Comprehensive experiments demonstrate that NanoSNP outperforms Clair, Pepper-DeepVariant and NanoCaller in identifying SNPs on low-coverage Nanopore sequencing data, including the difficult-to-map regions and major histocompatibility complex regions in the human genome. NanoSNP is comparable to Clair3 when the coverage exceeds 16×. https://github.com/huangnengCSU/NanoSNP.git. Supplementary data are available at Bioinformatics online.

A method of large DNA fragment enrichment for nanopore sequencing in region 22q11.2.

Background: 22q11.2 deletion syndrome (22q11.2DS) is a disorder caused when a small part of chromosome 22 is missing. Diagnosis is currently established by the identification of a heterozygous deletion at chromosome 22q11.2 through chromosomal microarray analysis or other genomic analyses. However, more accurate identification of the breakpoint contributes to a clearer understanding of the 22q11.2 deletion syndrome. Methods: In this study, we present a feasible nanopore sequencing method of 22q11.2 deletion. This DNA enrichment method-region-specific amplification (RSA)-is able to analyze the 22q11.2 deletion by specific amplification of an approximately 1-Mb region where the breakpoint might exist. RSA introduces universal primers into the target region DNA by a Y-shaped adaptor ligation and a single primer extension. The enriched products, completed by amplification with universal primers, are then processed by standard ONT ligation sequencing protocols. Results: RSA is able to deliver adequate coverage (>98%) and comparable long reads (average length >1Kb) throughout the 22q11.2 region. The long nanopore sequencing reads, derived from three umbilical cord blood samples, have facilitated the identification of the breakpoint of the 22q11.2 deletion, as well as by Sanger sequencing. Conclusion: The Oxford Nanopore MinION sequencer can use RSA to sequence the target region 22q11.2; this method could also be used for other hard-to-sequence parts of the genome.

De novo genome assembly and annotation of Holothuria scabra (Jaeger, 1833) from nanopore sequencing reads.

Holothuria scabra is a costly gourmet and traditional Chinese tonic medicine. However, the lack of high-quality genome information hinders the genetic, phylogenetic, and bioactivator researches. To construct high-quality genomic data of H. scabra and conduct genome-wide phylogenetic analysis. The whole genome of a male H. scabra was sequenced based on Nanopore MinION platform, and the sequence was assembled by wtdbg2. Transcriptome sequencing was used to aid the gene annotation. Repeat sequences, non-coding RNA, pseudogene and gene functional annotation were analyzed. 750 single-copy gene families from ten species were applied to construct phylogenetic tree for evolutionary analysis by using the ML method. The H. scabra genome of 1.18Gb (N50 = 1557,492bp) with 500.42Mb of putative repetitive sequences was assembled from a male H. scabra individual, and 16,642 protein-coding genes, 951 pseudogenes, 1791 motifs and 45,400 domains from the generated assembly were identified. The divergence time between H. scabra and its ancestor was estimated approximately 192.6 million years ago. H. scabra and A. japonicas joined together while sea urchin and sea star diverged about 440 Mya ago. Some key genes involved in notochord and gill slits development, skeleton degeneration and nervous system, as well as homeobox genes differ between H. scabra and Apostichopus japonicas. We report the first whole genome of H. scabra with expectation that this will be a valuable resource for genetic, phylogenetic, breading, molecular biology and bioactivator studies of sea cucumbers and other invertebrates.

A Pipeline NanoTRF as a New Tool for De Novo Satellite DNA Identification in the Raw Nanopore Sequencing Reads of Plant Genomes.

High-copy tandemly organized repeats (TRs), or satellite DNA, is an important but still enigmatic component of eukaryotic genomes. TRs comprise arrays of multi-copy and highly similar tandem repeats, which makes the elucidation of TRs a very challenging task. Oxford Nanopore sequencing data provide a valuable source of information on TR organization at the single molecule level. However, bioinformatics tools for de novo identification of TRs in raw Nanopore data have not been reported so far. We developed NanoTRF, a new python pipeline for TR repeat identification, characterization and consensus monomer sequence assembly. This new pipeline requires only a raw Nanopore read file from low-depth (<1×) genome sequencing. The program generates an informative html report and figures on TR genome abundance, monomer sequence and monomer length. In addition, NanoTRF performs annotation of transposable elements (TEs) sequences within or near satDNA arrays, and the information can be used to elucidate how TR–TE co-evolve in the genome. Moreover, we validated by FISH that the NanoTRF report is useful for the evaluation of TR chromosome organization—clustered or dispersed. Our findings showed that NanoTRF is a robust method for the de novo identification of satellite repeats in raw Nanopore data without prior read assembly. The obtained sequences can be used in many downstream analyses including genome assembly assistance and gap estimation, chromosome mapping and cytogenetic marker development.

Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species “L. swaminathanii”

Recently, a new Listeria species, “Listeria swaminathanii”, was proposed. Here, we phenotypically and genotypically characterize two additional strains that were previously obtained from soil samples and compare the results to the type strain. Complete genomes for both strains were assembled from hybrid Illumina and Nanopore sequencing reads and annotated. Further genomic analysis including average nucleotide identity (ANI) and detection of mobile genetic elements and genes of interest (e.g., virulence-associated) were conducted. The strains showed 98.7–98.8% ANI with the type strain. The UTK C1-0015 genome contained a partial monocin locus and a plasmid, while the UTK C1-0024 genome contained a full monocin locus and a prophage. Phenotypic characterization consistent with those performed on the proposed type strain was conducted to assess consistency of phenotypes across a greater diversity of the proposed species (n = 3 instead of n = 1). Only a few findings were notably different from those of the type strain, such as catalase activity, glycerol metabolism, starch metabolism, and growth at 41 °C. This study further expands our understanding of this newly proposed sensu stricto Listeria species.

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays.

Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionize the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the etiology of complex disease.