Reveal the Power of the 6-Base Genome

Reveal the Power of the 6-Base Genome From a Single DNA Molecule Liquid biopsy Cancer research Neurodegenerative disease Aging Precision medicine Mapping the role of 5mC and 5hmC The methylated bases 5mC and 5hmC are subject to dynamic regulation and can be influenced by various environmental factors, developmental stages and disease states.2 5mC and 5hmC modifications play critical roles in gene expression regulation in a cell-type-specific manner. Differentiating between them provides insight into the dynamic nature of DNA methylation and its impact on: • Closed chromatin • Repression of gene expression • Stable regional silencing • Euchromatin vs heterochromatin Methylation • Opening chromatin • Enhancing gene expression • Gene activation Hydroxymethylation Gene expression Cellular growth Disease development Conventional versus 6-base analysis The conventional 4-base approach sequences the DNA bases A, T, G and C. Adding the detection of 5mC and 5hmC creates the the 6-base genome. This approach provides a comprehensive view of genetic and epigenetic information in a single DNA fragment. Detailed insight into the interplay between genetic information and 5mC and 5hmC epigenetic markers can now be gathered using a single sample to provide vast amounts of biologically relevant data. Building predictive models with the 6-base genome As 5mC and 5hmC modifications have opposing functional roles, distinguishing between these two states allows researchers to develop predictive models. These models can help to determine gene expression patterns, establish chromatin accessibility and identify disease susceptibility. Key applications in translational research Combinatorial genetic and epigenetic insight also unlocks new avenues of exploration across a range of applications.  What you can get:  Sequence of the genome  Point mutations, insertions, deletions  Known variants associated with disease  What you can't get:  DNA modifications  Gene expression information  Environmental changes  What you can get:  Limited 4-base and modified cytosine information  Limited 4-base and modified cytosine information  What you can't get:  Differentiated methylation states, conferred by 5mC and 5hmC, that have opposing functionalities for gene expression and chromatin status • Somatic vs germline mutations • Methylation status of control elements • Differentially methylated regions (DMR) • Variant associated methylation (VAM) and hydroxymethylation (VAhM) Multidimensional insights from a single sample Conventional 4-base approach Conventional DNA methylation sequencing Groundbreaking 6-base analysis 6-base genome - with duet multiomics solution evoC Predicting biological outcomes This predictive insight enables you to: 5ng duet assay duet software 6-base genome multiomics data Genomics, epigenomics, transcriptomic, multiomics data analysis Sequencing Gather complete information in a single workflow from a single DNA sample  Combined genome and methylome  Full genetic sequence including variants  Epigenetic sequence differentiating between 5mC and 5hmC duet bioinformatics pipeline 5hmC 5mc Deconvolution of DNA encoded epigenetics can be correlated with the dynamic state of the genome. 5hmC and 5mC information can be used to train machine learning models of chromatin dynamics and gene expression. Models can be used to classify enhancer states based on their 5mC and 5hmC levels only. • Explore how methylation and hydroxymethylation influence gene expression. • Discover sensitive multimodal biomarkers for disease. • Understand and track the biology through disease stages. Chromatin accessibility Gene expression levels Enhancer status Cancer research Liquid biopsy Neurodegenerative diseases • Identify intra-tumoral heterogeneity. • Discover novel biomarkers that elucidate cancer stage. • Analyze potential biomarkers for non-invasive, earlier cancer detection. • Explore the relationship between genetic variants and changes in DNA methylation. • Study underlying molecular mechanisms. • Provide insight into disease with no clear genetic cause. Unlock transformative insights with duet evoC Integrating information on the four canonical bases with 5mC and 5hmC epigenetic markers enables researchers to generate genomic, epigenomic, transcriptomic and multiomic data that can fuel cutting-edge developments in translational research. Traditional next-generation sequencing (NGS) techniques are limited to the four bases and therefore can’t capture epigenetic nuances. Conventional epigenetic profiling tools also fail to detect C-to-T mutations or distinguish 5-methylcytosine (5mC) from 5-hydroxymethylcytosine (5hmC) modifications. But novel technological advancements are enabling the simultaneous sequencing of genetic and epigenetic bases. This infographic explores the 6-base genome and how it enables the exploration of epigenetic mechanisms of gene regulation through methylation and hydroxymethylation. Now, researchers are able to obtain an accurate measurement of 5mC and 5hmC, integrated into local genetic context, with >95% of bases above Q30 and 50% with accuracy above Q40. By measuring multiple modes of biology from a single lowinput DNA sample in a single experiment, researchers can use the comprehensive data to build predictive models of gene expression, chromatin accessibility and enhancer status to better understand the biological mechanisms that link genotype to phenotype.1 Genetic analysis provides insight into the information stored in the genome which, with limited exceptions, is relatively stable throughout an individuals' lifespan. In contrast, epigenetic changes govern cell differentiation and regulate gene expression in a more dynamic way, that is susceptible to environmental influences and aging. However, until recently, simultaneously sequencing both types of information has been challenging. Cancer Cardiovascular disease Metabolic disorders Autoimmune disorders Genetics Sequence of 4 bases (A-T-C-G) determines sequence of amino acids in encoded proteins ? 5mC 5hmC Aligned 6-base genome. Multiomic data generation for analysis, BAM, VCF, BEDmethyl, ASM reports. 5 Ref genome Resolved reads Sequencing paired-end read generates sequence information of the original and copied strand on a single molecule. 3 Read resolution uses the original and copied information to correctly call all 4 bases, 5mC and 5hmC. 4 Original Resolved read (4 bp) + (5mC, 5hmC) Copy R1 R2 Hairpin technology creates a single molecule with a direct copy of the original information to prevent information loss and enable base pair alignment calling to reduce errors. The methylation copy step copies methylation from 5mC on the original strand to the copy strand. 1 Strand synthesis, copy methylation Original Copy R1 R2 C 5mC 5hmC Protection, deamination and PCRmethylated cytosine (5mC and 5hmC) are protected. All unprotected cytosines are deaminated to uracil and subsequently converted to thymine during PCR. 2 Protection, deamination, PCR, NGS R1 R2 References 1. Füllgrabe J, Gosal WS, Creed P, et al. Simultaneous sequencing of genetic and epigenetic bases in DNA. Nat Biotechnol. 2023;41:1457–1464. doi:10.1038/s41587-022-01652-0 2. Breiling A, Lyko F. Epigenetic regulatory functions of DNA modifications: 5-methylcytosine and beyond. Epigenetics & Chromatin. 2015;8:24. doi:10.1186/s13072-015-0016-6 5mC 5hmC T Which epigenetic changes drive cellular differentiation pathways?