Abstract Comprehensive tumor characterization increasingly requires both genomic and epigenomic information, yet current methylation profiling methods, such as bisulfite-based workflows, limit sensitivity and accuracy due to DNA degradation and reduced sequence complexity. To address these constraints, we developed an improved positive-conversion chemistry that directly converts methylated cytosines while preserving unmethylated ones to enable simultaneous detection of cytosine modifications and genetic variants from a single NGS library. This approach is designed to support oncology research applications where high accuracy from limited or damaged material, such as FFPE tissue and cell-free circulating tumor DNA (ctDNA), is essential. To assess performance, FFPE, fresh-frozen, and cfDNA samples were processed using an optimized oxidation–reduction workflow. Libraries were sequenced on standard short-read platforms, and methylation accuracy, F1 scores, CNV concordance, and tumor–normal methylation contrast were evaluated. Performance was compared to whole-genome sequencing (WGS) controls and conventional bisulfite and enzymatic methylation sequencing methods.The chemistry demonstrated efficient reduction of modified cytosines, enabling high-confidence methylation calling with low false positive rates. Sequence complexity was maintained, allowing robust SNV and CNV detection from the same library. FFPE samples exhibited improved CpG coverage, fewer sequence artifacts, and improved library complexity compared to enzymatic methylation sequencing. cfDNA libraries generated from as little as 1 ng yielded reliable global methylation profiles suitable for tissue-of-origin analyses. The nondestructive nature of the chemistry preserved cfDNA fragments, opening the door to fragmentomics analyses. In tumor–normal comparisons, there was clear resolution of differentially methylated regions (DMRs), supporting more precise identification of tumor-specific epigenetic alterations. This enhanced positive-conversion chemistry enables unified genomic and epigenomic analysis from a single assay, making it highly suited for oncology research applications such as tumor classification, biomarker discovery, minimal residual disease (MRD) assessment, and liquid biopsy. Its gentle conditions, compatibility with degraded FFPE DNA and low-input cfDNA, and ability to capture both methylation and variants position it as a powerful tool for advancing translational research and precision oncology. Citation Format: Max Boeck, Jennifer Pavlica, Craig MARSHALL, Travis Sanders, Kristina Giorda, Martin Ranik, Eduard Casas, Thomas D. Harrison, Kailee Reed, Aaron Garnett, Doug Wendel, Brian Kudlow. Non-destructive methylation sequencing enables concurrent detection of genetic and epigenetic variation in FFPE and cfDNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1951.

Abstract In the pursuit of advancing multi-cancer early detection, the exploration of cell-free DNA (cfDNA) through methylation profiling has emerged as a promising avenue. However, the realization of its full potential is impeded by limitations inherent in conventional methodologies, notably the 'gold standard' bisulfite conversion methods. These approaches pose challenges such as DNA destruction and the generation of low-complexity sequences resulting from the conversion of unmethylated cytosines to thymine. TET Assisted Pyridine-Borane Sequencing (TAPS) holds the potential as a non-destructive, rapid, and scalable alternative for base-level methylation analysis. In this context, we introduce TET Assisted Pyridine-Borane Sequencing version 1.0 (TAPS v1.0), showcasing advancements to the technology originally introduced by Schuster-Böckler, Song, and their collaborators. TAPS v1.0 transforms 5mC into dihydrouracil (DHU) through a series of oxidation and reduction reactions. Ultimately, DHU is recognized as dU during PCR resulting in C-to-T conversions at 5mC sites, leaving unmethylated cytosines, the majority of cytosines, unchanged. The nondestructive nature of 5mC conversion yields significantly higher library yield and complexity compared to BS-seq. By leveraging Watchmaker's proprietary polymerase evolution platform, a DNA polymerase tolerant to DHU was selected for library amplification post DHU conversion, enabling unimpeded detection of 5mC, particularly in densely packed CpG repeats. Coupled with an optimized End Repair and A-tailing chemistry preceding TAPS v1.0, clinically relevant cfDNA samples of low quality and/or quantity can be analyzed in methylation detection pipelines. To evaluate the performance of TAPS v1.0, human genomic DNA with control spike-ins featuring varying methylation statuses were employed. TAPS v1.0 exhibited significantly higher final library yield and greater complexity compared to BS-seq. Similar outcomes were observed in clinically relevant cfDNA samples. Citation Format: Josh Haimes, Craig Marshall, Travis Sanders, Sam Vogel, Martin Ranik, Eduard Casas, Doug Wendel, Leo Karamanof, Bjarne Faurholme, Abre de Beer, Lee French, Ross Wadsworth, Kristina Giordia, Eric van der Walt, Brian Kudlow. Advancing methylation analysis: TAPS v1.0: A fast, scalable, and non-destructive sequencing method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7025.

Abstract Gene fusions due to chromosomal rearrangement, duplication, or deletion can be important drivers of cancer. Detection of gene fusions plays a valuable role in selecting targeted therapies. A widely used method for detecting fusion transcripts is targeted RNA sequencing, which focuses sequencing reads on known fusions but does not allow for the detection of novel fusions. Whole transcriptome analysis (WTA), where the entire transcriptome is interrogated, offers an opportunity to detect both known and novel fusions. Herein we examine the effect of sample quality, input mass, increased reverse transcriptase (RT) and insert size on fusion calling. Libraries are prepared using the Watchmaker RNA Library Preparation Kit with or without Polaris Depletion and, for target enrichment, the Twist Exome 2.0 panel prior to paired end 2 × 150 Illumina sequencing. First, control samples, specifically Seraseq Fusion RNA v4 high quality and FFPE samples, are used to determine if any of the parameters interrogated increase the number of breakpoint spanning reads both in a WTA and targeted sequencing context. Once established, the optimized workflow is applied to real FFPE samples. Targeted sequencing results in a higher number of breakpoint spanning reads, compared to WTA, when controlling for sequencing depth, resulting in a higher number of reads supporting each fusion call. Longer insert sizes, achieved by modified SPRI ratios, maintain library complexity and may enhance fusion calling. Taken together, these data demonstrate an optimized protocol for fusion calling from FFPE and demonstrate the utility of both WTA and targeted sequencing for fusion detection. Citation Format: Giulia Corbet, Josh Haimes, Travis Sanders, Martin Ranik, Thomas Harrison, Eduard Casas, Kailee Reed, Doug Wendel, Kristina Giorda, Ariele Hanek, Jen Pavlica, Brian Kudlow. Optimized RNA Seq library preparation for fusion calling from FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 321.

Clinical oncology heavily relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples for histology and molecular characterization. The chemical and physical modifications of nucleic acids introduced during fixation, storage and purification negatively impact molecular profiling and vary from sample-to-sample. Conventional sonication and ligation-based library preparation is considered the gold-standard approach for FFPE samples, but it is time-consuming and expensive. Importantly, these processes introduce artifacts that impact downstream analysis and interpretation (Haile et al., 2019). We have developed a novel fragmentation chemistry that virtually eliminates hairpin artifacts, achieving levels on par with non-FFPE control samples. Our fragmentation method is highly scalable, exhibits minimal sequence bias, and reduces cost and workflow inefficiencies associated with mechanical shearing. In this study, we developed a unified method for library preparation from FFPE samples which produces similar insert sizes across variable input mass and quality of FFPE samples. We carefully evaluated the performance of this method relative to a sonication-based approach employing the KAPA HyperPrep kit. Libraries were constructed from 50 to 200 ng of FFPE DNA, inputs typically used for NGS, ranging from low-to high-quality as assessed using a qPCR-based method and DNA integrity (DIN). Targeted sequencing was performed using a 37 kb custom oncology hybridization capture panel to investigate molecular complexity. Our workflow virtually eliminated hairpin artifacts that were present in up to 4.5% of reads in sonication-based libraries. Soft-clipping was also 3- to 7-fold lower in libraries prepared with the Watchmaker kit relative to sonicated DNA libraries, improving overall sequencing economy. Furthermore, the mean target coverage achieved with the Watchmaker kit was comparable to or higher than sonication libraries using the same input mass. Because input masses were normalized post-shearing, which typically results in 20-40% sample loss, coverage with our approach is significantly higher relative to sonication, if normalizing to pre-sheared DNA input. Watchmaker DNA Library Preparation with Fragmentation enables high-quality DNA library preparation from damaged FFPE samples, producing high target coverage, uniform insert size, and minimizing sequencing artifacts to improve sensitivity and specificity. This approach is highly scalable and automatable, enabling various oncology applications. Citation Format: Giulia Corbet, Philip Benson, Kailee Reed, Skyler Mishkin, Thomas Harrison, Kristin Scott, Zane Jaafar, Kristina Giorda, Josh Haimes, Martin Ranik, Brian Kudlow. A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 215.

After a decade of NGS technology and workflow innovation, personalized medicine is starting to become a reality. DNA fragmentation is still a critical bottleneck during library preparation. Sonication methods have been the gold standard for consistent fragmentation and uniform GC coverage, but are associated with a high upfront investment, expensive consumables, and are prone to oxidative DNA damage. On the other hand, enzymatic fragmentation methods hold the potential to be scalable, automation-friendly workflows that minimize DNA damage. Yet, typical enzymatic fragmentation methods have been shown to introduce systematic hairpin artifacts, exhibit suboptimal uniformity, and have a narrow range of input mass compatibility. We have developed a novel enzymatic fragmentation-based library preparation technology which, together with our high-fidelity library amplification module, effectively overcomes many of the key limitations of related chemistries. Libraries generated using our workflow reduced chimeric reads and terminal hairpin artifacts 10-fold compared to other enzymatic methods, and reached comparable levels of mechanically sheared DNA controls. Furthermore, we observed consistently even coverage uniformity and low sequence-specific bias for human whole genome sequencing. Fragmentation was tested across a broad sample input range from 100 pg to 500 ng. Library insert sizes were highly tunable from 150 bp to 550 bp and consistent across the input titration. High quality libraries with minimal adapter dimer were prepared from as little as 1 picogram of gDNA by adjusting the post amplification clean up conditions. Taken together, this enzymatic fragmentation and library preparation workflow avoids library preparation artifacts that convolute variant calling, is highly scalable, and suitable for ultra-low input samples. Citation Format: Zane Jaafar, Josh Haimes, Thomas Harrison, Lindsay Peterkin, Martin Ranik, Kristin Scott, Brian A. Kudlow. A novel enzymatic fragmentation library preparation chemistry that greatly reduces sequencing artifacts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2288.

Microbial whole-genome sequencingFor Research Use Only.Not for use in diagnostic procedures.A novel, single-tube enzymatic fragmentation and library construction method enables fast turnaround times and improved data quality for microbial whole-genome sequencing Next-generation whole genome sequencing of microbes demands rapid, robust, and scalable library construction workflows, capable of generating high-quality sequence data across a wide range of genome sizes, complexities and genomic GC content.In this Application Note, we describe a streamlined library preparation method that results in minimal bias, high uniform coverage, and facilitates de novo assembly of microbial genomes.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology.

Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Lignocellulosic biomass from fast-growing plantation trees is composed of carbohydrate-rich materials deposited into plant cell walls in a coordinated manner during wood formation. The diversity and evolution of the transcriptional networks regulating this process have not been studied extensively. We investigated patterns of species-level nucleotide diversity in the promoters of cellulose synthase (CesA) genes from different Eucalyptus tree species and assessed the possible roles of DNA sequence polymorphism in the gain or loss of cis-elements harboured within the promoters. Promoter regions of three primary and three secondary cell wall-associated CesA genes were isolated from 13 Eucalyptus species and were analysed for nucleotide and cis-element diversity. Species-level nucleotide diversity (π) ranged from 0.014 to 0.068, and different CesA promoters exhibited distinct patterns of sequence conservation. A set of 22 putative cis-elements were mapped to the CesA promoters using in silico methods. Forty-two percent of the mapped cis-element occurrences contained singleton polymorphisms which resulted in either gain or loss of a cis-element in a particular Eucalyptus species. The promoters of Eucalyptus CesA genes contained regions that are highly conserved at the species (Eucalyptus) and genus (with Arabidopsis and Populus) level, suggesting the presence of regulatory modules imposing functional constraint on such regions. Nucleotide polymorphisms in the CesA promoters more frequently created new cis-element occurrences than disrupted existing cis-element occurrences, a process which may be important for the maintenance and evolution of cellulose gene regulation in plants.

Because cultivation of exotic woody ornamental plants has led to establishment of a number of invasive species, there is considerable interest in breeding methods to reduce the propensity for spread. We review progress in conventional breeding and transgenic biotechnology approaches to producing sterile forms of ornamental woody plants. Conventional forms of inducing sterility, including induction of polyploidy, interspecific hybridization, and mutagenesis, are generally inexpensive and can be applied to a diversity of species at low to moderate cost. They have also been shown to be capable of producing commercially successful cultivars. In contrast, despite a variety of highly promising and rapidly developing approaches using transgenic methods, the inability to efficiently regenerate and genetically transform most ornamental species makes application of these innovations highly problematic. Moreover, because of the fragmented pattern of ornamental nursery ownership, the numerous species and varieties used, and the high regulatory cost for permits to sell most types of transgenic varieties (even when their environmental risk of spread has been reduced by sterility), application of transgenic methods is largely infeasible. A combination of fundamental regulatory reform and expanded biological research on generalized transformation and sterility methods is needed to overcome these barriers.

Background Cellulose is an important biopolymer produced by all plants and is used in a number of different industries, including for pulp and paper production. Cellulose is deposited into the plant cell wall by a large membranebound protein complex, which is composed of different cellulose synthase (CESA) proteins. The cellulose content and pattern of deposition in plant cell walls is highly variable depending on the function of the cell. All plant cells have a thin primary cell wall, but a number of plant cell types, including xylem cells, also deposit a secondary cell wall to give these tissues mechanical strength required to perform their function. Different cellulose synthase (CesA) genes have been shown to be involved in the deposition of primary and secondary walls. In Arabidopsis, three CesA (AtCesA4, 7 and 8) genes have consistently been associated with cells depositing secondary cell walls, while a different set of CesA genes have been shown to function during primary cell wall formation [Reviewed in 1]. These findings have been mirrored by studies of CesA gene orthologs in Populus and Eucalyptus[2-4]. While there have been a number of studies on CesA genes and their functions, much less is known about the regulation of these genes. In a previous study, we investigated the promoters of CesA genes involved in primary and secondary cell wall formation by performing a phylogenetic footprinting analysis to identify cis-elements conserved in the promoters from orthologous Arabidopsis, Populus and Eucalyptus cellulose synthase genes [5]. We identified a number of putative cis-regulatory elements that may play a role in the regulation of cellulose biosynthesis during primary and secondary cell wall formation. In the current study our aim is to further validate the ciselements identified in the CesA gene promoters by investigating their conservation across different Eucalyptus species and to determine the regulatory function of these promoter regions and the proteins which bind to them.

Background Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform in vitro. Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.