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, 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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Organic Extraction of RNA From Animal and Plant Tissues Without Phase Separation
Stanislav Forman , Danice Anne A. Cabaya , Xi Yu Jia

Organic acid-guanidinium-phenol based extraction is widely implemented for most plant and animal RNA purification protocols. This organic extraction method effectively inactivates nucleases and infectious agents and allows for adequate sample storage and stabilization. However, limitations of existing organic extraction methods become apparent with high-throughput processing and the handling of small volume inputs. In both cases, the requirement of phase separation can affect both the yield and purity of RNA. Here we present an alternative acid-guanidinium-phenol based procedure that effectively bypasses phase separation/precipitation steps with a spin column and specially designed washes. This helps to eliminate problems attributed to phenol carryover that is often associated with conventional organic extraction methods. This new “Direct-zol™” procedure maximizes total RNA recovery, including small RNAs, without the need for a carrier. For tough-to-lyse plant and animal tissue the Direct-zol™ procedure can be combined with unique BashingBead™ and OneStep inhibitor removal technologies for unparalleled yields and complete removal of polyphenolic inhibitors to RT-PCR, respectively. The Direct-zol™ procedure delivers DNA-free RNA that is ideal for RT-PCR, RNA-seq (expression profiling), hybridization, etc.

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