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.

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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Bisulfite Treatment of DNA: Perfecting the Deamination of Cytosine Into Uracil
Seth A. Ruga, Michael Karberg, Xi-Yu Jia, Marc E. Van Eden

Sodium bisulfite can deaminate “convert” cytosine in DNA into uracil, but does not affect 5-methylcytosine. Bisulfite treatment of DNA is a prerequisite for DNA methylation analysis for many epigenetics-based studies involving methylation profiling and the quantification of methylation status. However, analytical procedures involving bisulfite treated DNA are often subject to variability due to DNA degradation, incomplete conversion, and/or low yields of DNA. We have systematically investigated the procedure of bisulfite treatment of DNA paying particular attention to the chemistries involved in the process and to conversion rates in an effort to limit variability between samples and to improve upon conventional methods. We found conventional bisulfite DNA conversion chemistries could be improved such that increased C to U conversion efficiencies could be obtained without the levels of DNA degradation typically resulting from incubation of reaction mixtures at high temperature and nonphysiological pH. Essential to this process was prohibiting the occurrence of "over-conversion" of 5-methylcytosine into uracil that can occur in some situations and reaction conditions. We found the bisulfite conversion process could be simplified and the variability between treatments kept to a minimum by coupling heat denaturation with the bisulfite conversion process and by using in-column desulphonation to clean and purify the converted DNA. This new method was found to yield an average of > 80% recovery of input DNA with > 99% C to U conversion. The method has been specifically designed to accommodate (in addition to purified DNA) biological fluids, cells, or tissue directly as the input material. This makes its application for FFPE and LCM-derived samples particularly well suited.

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DNA Methylation Biomarkers for Early Bladder Cancer Detection and Treatment Response Monitoring
Daneshmand S., Seyedian S.L., Ghodossipour S., Hamed A., Roberts S., Ghoreifi A., Basin M.F., Simin H., Piatti P., Bhasin J., Jara B., Sanossian L., Chew Y.C., Djaladat H., Schuckman A., Bhanvadia S., Liang G.

Cystoscopy and urine cytology are routinely employed for the detection and monitoring of bladder cancer. Diagnostic accuracy of FDA approved urine-based tests is suboptimal and provide no, or limited, prognostic value. Herein, we evaluate the diagnostic values of the Bladder CARE Assay, a urine-based DNA methylation test recently granted with FDA Breakthrough Device Designation, in: 1) Detecting bladder cancer prior to current standard of care. 2) Identifying response to anticancer therapies. 3) Predicting tumor recurrence based on residual signal after TURBT

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