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 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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Correlation Between Histone Modifications and 5-Hydroxymethylation in Human Brain
Yap Ching Chew, Adam Petterson, Tzu Hung Chung, Xueguang Sun, and Xi Yu Jia

Histone tails that are exposed at the nucleosomal surface are targets for covalent modifications such as acetylation, methylation, and phosphorylation. These post-translational modifications play crucial roles in the regulation of chromatin structure and maintenance of genomic stability. DNA methylation of CpG dinucleotides is another epigenetic event involved in gene regulation. Recent studies demonstrated that 5- methylcytosine (5-mC) can be further modified to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (Tet) family proteins. The balance between 5-mC and 5-hmC in the genome is a critical step for regulating gene expression to maintain cellular functions. 5-hmC is particularly enriched in the brain when compared to other tissues. However, the biological mechanisms linking histone modifications and 5-hmC in human brain and other tissues have not yet been established. Reduced Representation Hydroxymethylation Profiling (RRHP) is a method that combines whole-genome library preparation with selective adapterization and Next-Gen sequencing. Using RRHP, a new technique that map 5-hmC at single-nucleotide resolution in a strand-specific fashion, we analyzed the 5-hmC profiles of human male cerebellum. The correlations between 5-hmC and histone modifications are assessed using functional annotation analysis. We observed that 5-hmC enriched regions overlapped with H3K4me3 (a euchromatin mark)- associated regions, accounting for more than 50% of the overlap, whereas, only 25% of 5-hmC sites overlapped with H3K27me3 (a transcription repressive mark)-associated regions. Additionally, we also observed 12% of 5-hmC overlapped with bivalent domains which consisting both H3K4me3 and H3K27me3 marks. Taken together, our newly developed technique, RRHP provides new insight into the relationship between 5-hmC and histone modifications, and will be a powerful tool for future studies on the diverse regulatory roles associated with 5-hmC and histone modifications.

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