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.

Quantitative Digital PCR Analysis of Cancer Gene Promoter Methylation Using Low Amounts of Input DNA
Paolo Piatti, Alireza Ghoreifi, Sanam Seyedian, Yap Ching Chew, Benjamin Jara, Lucy Sanossian, Jeffrey Bhasin, Michael Basin, Taikun Yamada, Gerhard Fuchs, Sumeet Bhanvadia, Rene Sotelo, Andrew Hung, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, Hooman Djalada

Silencing of tumor suppressor and tumor-related gene transcription by hypermethylation at promoter CpG motifs is a significant mechanism at work in human tumorgenesis. Various methods have been developed to analyze DNA methylation levels, both across the genome and at specific loci, in order to discover and interrogate disease relevant loci for methylation-based transcriptional control. Methods able to quantitatively measure differences in DNA methylation between normal and cancer cells provide promising sources for biomarker identification and assessment. Zymo Research's OneStep qMethyl™ Kit is used for the detection of region-specific DNA methylation via the selective amplification of methylated cytosines after digestion with methylation-sensitive restriction enzymes. RainDance Technologies' RainDrop™ dPCR system is a highly sensitive tool for precise quantification of nucleic acids using probe-based qPCR reagents. RainDrop offers unique analytical advantages for clinical diagnostics due to its exceptionally high sensitivity for absolute quantification and capability to multiplex assays, using a wide dynamic range of input DNA in a contamination-free and simple workflow. The combination of both technologies enables a novel, fast, and robust method for quantitative methylation analysis of small amounts of DNA. Here we report on the use of Zymo's methylation assay and RainDrop digital PCR counting to enable quantitative measurements of DNA methylation at specific genomic loci using small amounts of non-bisulfite converted DNA. MGMT promoter methylation measurements are used to illustrate the method's robust quantification of small percent methylation changes using low amounts of input DNA in a single-plex and duplex assay format (duplex RAB25-VIC and MGMT-FAM probes). Duplex assays using a methylation-independent reference assay and either RARB or CCND2 are used to assess promoter methylation in breast cancer tumor and adjacent normal tissue samples showing stage-specific differential methylation.

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Rapid Enzymatic DNA Degradation for Quantitation of 5-Methylcytosine and 5-Hydroxymethylcytosine
James L. Yen & Xi Yu Jia

Modification of DNA, namely 5- methylcytosine, has been recognized to be one of the dominant phenomenon in the field of epigenetics. Fluctuations in global DNA methylation levels have implications in development, cancer, and aging. Therefore methods for precise quantification of global DNA methylation (i.e. - HPLC and LC/MS) are powerful tools to gain a better understanding in these areas. To address the need for a rapid and convenient sample preparation method for global methylation quantitation, we have developed a one hour, one-step enzymatic procedure for DNA degradation with DNA Degradase™ and DNA Degradase Plus™. DNA Degradase™ and DNA Degradase Plus™ degrade DNA to single nucleotides and nucleosides, respectively. Nucleotides are easily quantitated by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC), while nucleosides (lacking a charged phosphate) are ideal for quantitation by mass spectrometry (LC/MS). Furthermore, we have validated DNA Degradase™ and DNA Degradase Plus™ by HPLC nand LC/MS, respectively. Sampling a range of biological sources, DNA Degradase Plus™ coupled with LC/MS has proven to be a powerful method for detection and quantification of 5-methylcytosine as well as 5-hydroxymethylcytosine.

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Read-Level Methylation Pattern Extraction for High-Multiplex Large-Scale Targeted NGS Assay
Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia

The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with highmultiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true

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Take the Lab to the Field With a Portable Sample Processor and DNA Stabilization Reagent
Nikolas M. Isely, Steven Wong, Marc E. Van Eden, and Xi Yu Jia

With the increased focus of research on the effects of global climate change, natural disasters, and anthropogenic perturbations on the environment it is becoming more important to collect and process samples for molecular analysis in remote locations. Working in the field presents several challenges including sample processing and nucleic acid stabilization. Historically it has been necessary to transport bulky and expensive equipment into the field to process and preserve samples. To tackle these challenges Zymo Research has developed a hand-held, portable bead beating device, the Xpedition™ Sample Processor (XSP). When the XSP is used in conjunction with the specially developed Xpedition™ Lysis and Stabilization Solution, DNA is preserved in complex and harsh lysates without the need for refrigeration for greater than 7 months. DNA isolated from fungus, soil, plants, and animal tissue using the Xpedition™ Environmental Kits is ideal for use in modern molecular analysis techniques like qPCR, metagenomic analysis, and DNA barcoding. Here we present data showing the preservation and stabilization of DNA at room temperature for extended periods of time in complex lysates.

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Universal rRNA Depletion for Transcriptome Analysis of Any Organism with a Streamlined, Autolaunch Bioinformatics Platform
Yi Xu, Kate V. Kuntz, Jeffrey M. Piña, Zhenfeng Liu

Depletion of ribosomal RNA (rRNA) is commonly adopted in total RNA-seq experiments to maximize the sequencing efficiency given its overabundance in RNA samples.1 Most existing rRNA depletion strategies are probe-based and well-established for human samples and mouse/rat models. Research utilizing other models and non-model organisms often lacks a simple and reliable rRNA depletion solution. To address such a need, Zymo Research developed a novel probe-free rRNA depletion strategy, RiboFree Universal Depletion, which is integrated into a simplified library prep procedure to enable stranded, total RNA library preparation for any organism. Furthermore, Zymo Research deposited an RNA-Seq data analysis pipeline to a no-code platform called Aladdin (www.aladdin101.org) for researchers to complete essential bioinformatic analyses including differential gene expression and gene set enrichment analyses in a streamlined, point-and-click manner. The RiboFree technology and the Aladdin bioinformatics platform will greatly empower researchers from diverse backgrounds to make further impactful contributions.

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