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.

A Novel Sequencing Method for Genome-Wide Profiling of 5-Hydroxymethylcytosine With Single-Base Resolution
Darany Tan, TzuHung Chung, Xueguang Sun & Xi-Yu Jia

5-hydroxymethylcytosine (5hmC) is an epigenetic mark abundant in embryo stem cells and brain tissues. The exact biological functions of 5hmC are still under close investigation although several lines of evidence have indicated it could be involved in active DNA demethylation. Meanwhile, extensive studies have been carried out to determine its genomic distribution. A number of approaches have been developed using either affinity based enrichment, such as hMeDIP, that rely on antibody and other specific binding proteins to target 5hmC, or modified bisulfite sequencing, namely oxidative bisulfite sequencing (OxBS) and TET assisted bisulfite sequencing (TAB-sequencing). However, all those methods have limitations which hamper their application. For example, affinity based methods lack single base resolution while modified bisulfite sequencing methods require efficient chemical or enzymatic oxidation which cannot be easily achieved or guaranteed. As an alternative, we have developed a novel genome-wide sequencing method that utilizes an enzyme based modification approach coupled with bisulfite-sequencing for detecting 5hmC. This methodology allows quantification of 5hmC levels with single CpG resolution and can also be employed for locus-specific assays. Using this method, we were able to map and quantify 5hmC sites at the genomic scale for several different biological samples. This novel method can determine the exact location and abundance of 5hmC, which will facilitate our understanding of 5hmC in regulating gene expression in different biological contexts.

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A Urine-Based DNA Methylation Test to Detect Upper Tract Urothelial Carcinoma: Results from a Prospective Pilot Study
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 Djaladat

Upper Tract Urothelial Carcinoma (UTUC) is an uncommon, yet lethal malignancy of the urinary tract. Diagnosis and preoperative risk stratification of UTUC patients present distinct challenges given the limitations of currently available tools such as urine cytology, endoscopic biopsy, and cross-sectional imaging. Decision-making and prognosis of UTUC relies heavily on TNM stage and pathological grade, which are is not accurately available until RNU is performed.

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Accelerated Epigenetic Aging in Bladder Cancer Patients
Yap Ching Chew, Wei Guo, Xiaojing Yang, Paolo Piatti, Mingda Jin, Keith Booher, Michiko Suwoto, and Xi Yu Jia

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome-wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low-read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM (Simplified Whole-panel Amplification Reaction Method), for the accurate biological age determination. SWARM is flexible and low cost, requires relatively low DNA starting material, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM approach, we were able to analyze the methylation level of several hundreds of age-associated loci including the published Horvath Clock sites. Gender-specific age-predictive models were built using the elastic net regression of DNA methylation levels of the loci and chronological age of urine DNA samples of over 300 healthy subjects of 18 to 88 years old. Urine samples from bladder cancer patients exhibit significant age acceleration, with an average of >10 years. In brief, our gender-specific urine DNAge analysis is a tool for the precise biological aging quantification and can be used to address questions in aging and urinary tract health.

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Applications of Innovative Whole-Genome Bisulfite Sequencing (WGBS) for Cancer Biomarker Discovery in Plasma Liquid Biopsy
Caila Ruiz, Hanjun Kim, Ryan Yancey, Jeffrey Bhasin, Mingda Jin, Yi Xu, Xiaojing Yang, Larry Jia

Circulating cell-free DNA (cfDNA) has been reported to contain valuable genetic and epigenetic information for the diagnosis and prognosis of cancer. Studies have shown that the blood from cancer patients contains more tumor-derived cfDNA compared to the healthy controls. DNA methylation, the most well-studied epigenetic marker, has been validated as one of the key drivers in the development of many diseases including cancer. cfDNA methylation thus holds great potential to become a biomarker that will enable early detection of cancer. However, cfDNA is also a challenging input for library preparation as cfDNA is mostly highly fragmented, and the amount of cfDNA from blood plasma is comparatively low. Two classes of library preparation protocols were evaluated for profiling DNA methylation patterns at single nucleotide resolution from ultra-low amount of blood plasma cfDNA. These methods included a bisulfite conversion-based protocol and an enzymatic-based protocol. The bisulfite conversion reaction was optimized allowing a milder treatment for less nucleic acid damage. This bisulfite conversion-based protocol also contained a further optimized adapter ligation step, allowing a simpler procedure. Circulating cfDNA were extracted from blood plasma of a healthy control, a non-small cell lung cancer patient, and an adenocarcinoma lung cancer patient. 5-ng cfDNA was used as input and the libraries were successfully prepared using both methods. Each library was sequenced to 300 – 600 million read pairs at a read length of 150 bp, enabling a median coverage of 20X per detected CpG, and 86.8% of the 30 million CpG sites in human genome were covered by at least 1 read. Unique alignment rate among all libraries were about or above 80%. The comprehensive coverage of the CpG sites across the entire human genome illustrated that the optimized bisulfite conversion reaction was compatible with fragmented cfDNA, proving the satisfactory efficacy in this simple and classic method for DNA methylation profiling of such a challenging sample type. This opens the door to apply the streamlined and simple Whole-Genome Bisulfite Sequencing (WGBS) protocol for cfDNA methylation profiling, which in turn shall facilitate further understanding in cfDNA epigenetic variations and advance the development of cancer biomarkers from the non-invasive, liquid biopsies

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Automated Unbiased Metagenomic DNA Extraction for Long-Read Nanopore Sequencing
Joshua Tjokrosurjo, Angel Gonzalez, Kevin Lin, Shuiquan Tang, and Xi Yu Jia

The advantages of nanopore sequencing include the capacity to generate very long-reads with remarkable speed and portability, spanning tandem-repeat regions resolving ambiguity during genome assembly. However, extracting inhibitor-free HMW DNA suitable for long-read sequencing has always been a challenge due to DNA fragmentation and contaminant introduction. Furthermore, applying this to metagenomic samples increases difficulty as composition bias can be introduced during DNA extraction and shearing due to the presence of DNases. Here we present an automated HMW DNA extraction pipeline that combines magnetic bead-based DNA extraction with the Microlab STAR™. Magnetic bead chemistry allows for the retention of HMW DNA, cleaner elutions, and is amenable to high throughput liquid handling applications. Eluted DNA from the pipeline was sequenced using the MinION™ platform; achieving 1M reads, 8 Gb throughput, with average read-lengths of 8 kb, with over 100 kb recorded. Our results demonstrate that the extracted microbial profile closely matches the theoretical composition. Overall, we have developed an automated method for HMW DNA extraction that shows unbiased microbial lysis that is compatible with long-read nanopore sequencing

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