Scientific Posters

Zymo Research's scientific posters highlight the latest breakthroughs in molecular biology, showcasing innovations spanning from epigenetics to microbiomics. We are proud to present insights from our collaborations with leading scientists and institutions worldwide, with more contributions to the field of life science on the horizon.

Genome-Wide Human Brain DNA 5-hmC Profiling Using a Novel Sequence- and Strand-Specific Method
Xueguang Sun, Adam Petterson, Tzu Hung Chung, Xi Yu Jia, Pu Zhang

5-Hydroxymethylcytosine (5-hmC) is an epigenetic hallmark which has recently become central in mapping and sequencing work. While the exact function of this base is not fully understood, it is likely to regulate gene expression as a member of active DNA demethylation pathways. The levels of 5-hmC in genomic DNA vary significantly depending on the cell type, though the highest levels are found in cells of the central nervous system (CNS): These findings suggest importance of 5- hmC in gene regulation within the CNS. While several methods have been developed to profile 5-hmC at genomic scale, most are enrichment-based, utilize large amounts of genomic DNA input, and have relatively low resolution. Although efforts have been made to detect 5hmC at single-site resolution, the methods described to date still require several micrograms of DNA, require parallel or subtractive sequencing, and employ successive chemical treatments that degrade the DNA and hinder sequencing. By combining modification-sensitive restriction enzymes with massively parallel (“next-generation”) sequencing approaches, we developed a novel Reduced Representation Hydroxymethylation Profiling (RRHP) method for genome-wide 5-hmC mapping at single-site resolution from low (100 ng) DNA inputs. Importantly, the method can detect strand polarity of 5-hmC modifications, and also enables the direct identification of single nucleotide polymorphisms (SNPs) within sequencing reads. Due to the fragmentation approach, data can be directly compared with single-base DNA methylation data from Reduced Representation Bisulfite Sequencing (RRBS). Human brain 5-hmC mapping generated with this method, combined with DNA methylation profiling data, indicates unique distributions of 5-hmC modification: We confirm that several important neuronal loci, such as BDNF, NLGN2, CES1, and TAF1, demonstrate extensive 5-hmC modification. This new method of detection and mapping is a powerful tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in neurobiology and other diverse biological fields

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Genome-Wide Human Brain DNA 5-hmC Profiling Using a Novel Sequence- and Strand-Specific Method
Xueguang Sun, Adam Petterson, Tzu Hung Chung, Marc E. Van Eden, and Xi Yu Jia

5-Hydroxymethylcytosine (5-hmC) is an epigenetic hallmark rapidly gaining much interest within mapping and sequencing disciplines. While the precise role of 5-hmC is not fully understood, it is implicated in regulation of gene expression via active DNA demethylation pathways. Previous studies demonstrate that it plays a role in cell differentiation and carcinogenesis: Cells that are more stem- and progenitor-like have greatly reduced levels of 5-hmC compared with more differentiated cells. Similarly, tumor cells display less 5-hmC than their normal counterparts independent of either grade or stage, suggesting that global loss of 5-hmC may be an early event in carcinogenesis. Several methods have been described to profile 5-hmC at the genomic level: Most are enrichment-based via immunoprecipitation or other bioorthogonal labeling schemes, and several conversion methods have also been described that exploit selective oxidation. Here we employ a new method which combines modification-sensitive restriction enzymes with next-generation sequencing approaches to allow genome-wide 5-hmC mapping at single-site resolution in several families of carcinomas. This new method should provide a unique tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in carcinogenesis.

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Genomic Approach for DNA Methylation and Hydroxymethylation Analysis
M. Krispin, X. Sun, R. Leavitt, D. Butler, W. Pirovano, B. Reichert, T. Chung, E. Bacon, A. Petterson, M. Van Eden, X. Jia

DNA methylation and hydroxymethylation are some of the most important epigenetic modifications that can occur in the human genome. For instance, DNA methylation plays a vital role in the regulation of gene expression in normal cell development and aging, and also in the formation and progression of cancer and other diseases. Large scale identification of putative epigenetic biomarker candidates is now achievable with the ability to profile DNA methylation and hydroxymethylation at the genomic level. Once validated, specific biomarkers could be applied to clinical and molecular diagnostic fields. Due to the increased availability of Next-Gen sequencing technology, a number of new technologies have been developed for interrogating DNA methylation and hydroxymethylation at the genomic scale. Zymo Research has recently perfected sample prep and bioinformatics analysis as part of its new DNA Methylation and Hydroxymethylation Profiling Services. These epigenetic services combine next-generation sequencing with Zymo's well-established epigenetic technologies and innovative bioinformatics algorithms for the most streamlined, comprehensive genome scale data generation to date. With these new services, hundreds of epigenomic biomarker candidates can be discovered simultaneously. Furthermore, Zymo Research offers services for validation of biomarker candidates via targeted sequencing or qPCR. abstract Introduction

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Increasing Reliability of Microbiome Diagnostics of GI Disease by Sample Preservation of Stool and Automated Unbiased Nucleic Acid Extraction
Kevin Lin, Luigi Basilio, Shuiquan Tang, Stanislav Forman, Ryan Kemp, and Xi Yu Jia

Background: The gut microbiota has long been associated with GI diseases including, Crohn's disease, ulcerative colitis, and inflammatory bowel disease. Understanding the gut microbiota holds promise for earlier clinical diagnosis of these types of diseases. However, diagnostic success is predicated on accurate detection of microbes in the stool and oftentimes sample degradation or changes can occur due to improper storage which leads to biased results. To combat this, we evaluated a sample collection medium that preserves genetic profiles, inactivates pathogens, and is suitable for direct automated nucleic acid extraction. Methods: Human stool samples were subjected to storage in DNA/RNA Shield™ (preservation medium) versus unprotected samples at ambient temperatures and were also subjected to repeated freeze-thawing cycling from -80°C. After bead-beating homogenization, DNA was extracted using an automated microbiome workflow on a Tecan Fluent™. Microbial profiles were analyzed using 16S rRNA gene sequencing on Illumina® MiSeq™ targeting the V3-V4 region. Additionally, a mock microbial standard of various gram positive/negative bacteria and yeast species were used to test the performance of various extraction methods to determine efficiency of lysis. Results: Microbial composition in preserved stool was unchanged up to 1 month and was unaffected by freeze thaw (up to 10 cycles). Unprotected samples experienced a shift in microbial profiles in as little as one day with unaccounted microbial growth. Overall, there was a complete loss of Bacteroides and significant increase in Actinobacteria in as little as 5 freeze thaw cycles. A majority of extraction methods also revealed a bias toward gram negative species that portrayed a skewed representation of the microbiome. Conclusions: Microbial profiles remained consistent at ambient temperatures when stool was stored in DNA/RNA Shield™. Furthermore, the preservation medium facilitated nucleic acid extraction and was amenable for direct automated processing on various instrumentation and chemistries.

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OneStep qMethyl™ Panel: A Method To Indicate Pluripotency and Characterize Human Embryonic Stem Cells
Lam Nguyen, Jill Petrisko, Manuel Krispin, Xi-Yu Jia

Pluripotency is the ability of embryonic stem cells to differentiate into multiple cell types. Pluripotent cells have epigenetic signatures that reflect their ability to generate multiple cell types. Different DNA methylation patterns in gene regions vary between pluripotent and differentiated cells as a result of processes such as development, carcinogenesis, genomic imprinting disorders, and cell reprogramming. In human pluripotent cells, gene promoter regions in the NANOG, RAB25, and PTPN6 genes have been shown to maintain low levels of DNA methylation compared to differentiated cell types. Conversely, gene promoter regions of MGMT, GBP3, and LYST have been shown to maintain high levels of methylation in pluripotent cells compared to differentiated cell types. Here we present a simple, straightforward, and bisulfite-free procedure for rapid, DNA methylation assessment for the above mentioned genes.

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