Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased transmission risks in downstream environments. Advancements were also made toward standardizing methods for the generation of globally representative and comparable metagenomic- and culture-based AR monitoring data via two comprehensive and critical literature reviews. The first review provides guidance in next-generation sequencing (NGS) studies of environmental AR, proposing a framework for experimental controls, adequate sequencing depths, appropriate use of public databases, and the derivation of datatypes that are conducive for risk assessment. The second review focuses on antibiotic-resistant Enterococcus spp. as robust monitoring targets and an attractive alternative to more widely adopted Gram-negative organisms, while proposing workflows that generate universally equivalent datatypes. Finally, quantitative metagenomic (qMeta) techniques were benchmarked using internal reference standards for high-throughput quantification of ARGs with statistical reproducibility. / Doctor of Philosophy / Antimicrobials have contributed to the reduction of infectious diseases in humans and animals since the early 20th century, increasing productivity and saving countless lives. However, their industrial-scale application across human, animal, and agricultural sectors over the last several decades, especially the use of antibiotics, have engendered the proliferation of antibiotic resistance (AR). AR occurs when changes in bacteria cause the drugs used to treat infections to become less effective and has become one of the leading public health threats of the 21st century. The global spread of AR through the transmission and evolution of antibiotic resistant bacteria (ARB; known colloquially as "superbugs") and antibiotic resistance genes (ARGs) across the One Health continuum (i.e., humans, animals, and the environment) is resulting in increased hospitalization, length of hospital stays, suffering, death, and overall health-care associated costs globally. This dissertation demonstrates the use of metagenomics, the sequencing of all genetic material (e.g., DNA) recovered from a microbial community, for the comprehensive monitoring of ARB and ARGs in aquatic environments, a key pathway for the dissemination of AR into and out of human populations. In order to impede the proliferation of AR, surveillance systems are currently in place to track the spread and evolution of ARB and ARGs in humans and livestock, as well as agri-food sectors. However, the surveillance in natural and built environments (i.e., rivers and domestic sewage) has significantly lagged due to the lack of standard monitoring targets and methodologies. It is also a goal of this dissertation to suggest guidance for the collection of metagenomic- and culture-based AR monitoring data to generate universally comparable results that can be included in centralized databases. Riverine systems are ideal models for tracking input of antibiotic resistance to the natural environment by human activity. After Hurricane-Maria, many of Puerto Rico's wastewater treatment plants (WWTPs) went offline, discharging raw sewage to local surface waters. In a cross-sectional study of watersheds impacted by WWTPs, the abundance of ARGs was directly correlated to increases in local population density. Also, highly mobile and clinically-relevant ARGs were found directly downstream of WWTPs across the island. We found that many of these ARGs corresponded well to forms AR endemic to the region. WWTPs are the primary engineering controls put in place to curb the spread of human and animal waste streams and can help to reduce AR. An international transect of conventional activated sludge WWTPs representing US/Europe and Asia were sampled to garner a mechanistic understanding of the fate or ARGs through treatment. Although WWTPs remove total bacteria, human fecal indicators, and much of the abundance of ARGs, mobile and clinically-relevant ARGs are discharged around the world in large quantities. Consideration is needed in certain regions of iv the world where the managing of human waste streams is the first line of defense against the dissemination of resistance to local communities. Two comprehensive critical literature reviews were conducted to evaluate the various methodologies for generating and analyzing metagenomic- and culture-based AR monitoring data. These reviews address the need for experimental rigor and disclosure of extensive metadata for inclusion in future, centralized databases. The articles further provide guidance with respect to universally comparable datatypes and efficient workflows that will aid in the scale-up of the collection of environmental monitoring data within a global surveillance framework. Finally, a study was conducted to benchmark the use of internal DNA reference standards for the absolute quantification of ARGs (i.e., on a ARG copy per volume of sample basis). The statistical framework for ARG detection and its implications for wastewater-based surveillance systems of AR are also discussed.

Antimicrobial resistance (AMR) threatens our modern standard of living with the potential return to a pre-antibiotic condition where deadly infections are no longer treatable. Wastewater treatment plants (WWTPs) are vital components in water sanitation infrastructure and are now also being recognized as valuable monitoring points for antibiotics, antibiotic resistant bacteria (ARB), and antibiotic resistance genes (ARGs) disposed of or excreted by human populations. Hospital waste water is of special interest as a potential focused monitoring point and in general research is needed to establish the benefits of both on-site and community-scale wastewater treatment as important barriers to the disseminators of ARGs into the environment. The research aims described herein examine these components of wastewater treatment and how they relate to AMR indicators identified through metagenomic sequencing. Through monitoring of local WWTPs, it was found that AMR indicators shifted over time and in relation to human behavior that changed due to the COVID-19 pandemic. Hospital wastewater did not measurably impact the microbiome during simulated activated sludge wastewater treatment according to broad-scale metagenomic ARG profiling; however, some clinically-relevant ARGs escaped treatment. Lastly, a study of a transect of WWTPs indicated impacts on the abundance of certain ARGs in downstream riverine receiving environments. Nonetheless, there appeared to be a number of other factors at play, and upstream and downstream resistomes tended to remain similar, calling for further research to delineate impacts of various wastewaters and treatments on ARGs in affected aquatic environments. / Doctor of Philosophy / Antimicrobial resistance (AMR) occurs when bacteria, viruses, and fungi are able to survive in the presence of antibiotics because they carry antibiotic resistance genes (ARGs) encoded in their DNA. AMR is a major public health concern as it makes it so that antibiotics are no longer effective against potentially deadly infections. Wastewater treatment plants (WWTPs) are being discovered as a hub of opportunity for monitoring potential AMR problems in a community. WWTPs receive sewage from homes and various industries. This sewage contains rich information for researchers to examine in terms of which antibiotics, bacteria, and ARGs are circulating in the community. This makes it possible to find out which antibiotics are being consumed in the community and which ARGs might be prevalent. The purpose of this research was to better understand both how WWTPs can be used as monitoring points for AMR and how they can be improved to help reduce ARGs emitted to rivers and streams where treated water is discharged. It was found that the types of ARGs prevalent in wastewater changed over time, especially during the COVID-19 pandemic as people worked from home and changed habits regarding doctors' visits, which impacted antibiotic use. Hospital sewage was studied as a useful indicator of pathogens and ARGs that are challenging a community and also the antibiotics being used. This research explored what happened to ARGs during the treatment of domestic (i.e., from people's homes) wastewater along with hospital wastewater and found that hospital wastewater introduced some ARGs that are typically found in clinical settings, but did not negatively impact the overall wastewater treatment process. Finally, the impact that WWTPs have on rivers to which treated water is discharged was explored. The results indicated that certain ARGs were elevated downstream of the WWTPs. However, when examining all ARGs together, no major shifts due to the treated wastewater were apparent.

The main focus of this research was the development of microfluidic technology for ultrasensitive and fast molecular analysis of cells. Chromatin immunoprecipitation (ChIP) assay followed by next generation sequencing serves as the primary technique to characterize the genomic locations associated with histone modifications. However, conventional ChIP-seq assay requires large numbers of cells. We demonstrate a novel microfluidics-based ChIP-seq assay which dramatically reduced the required cell number. Coupled with next generation sequencing, the assay permitted the analysis of histone modifications at the whole genome from as few as ~100 cells. Using the same device, we demonstrated that MeDIP-seq with tiny amount of DNA (<5ng) generated high quality genome-wide profiles of DNA methylation. Off-chip sonication often leads to sample loss due to multiple tube transferring. In addition, conventional sonicators are not able to manipulate samples with small volume. We developed a novel microfluidic sonicator, which is able to achieve on-chip DNA/chromatin shearing into ideal fragment size (100~600bp) for both chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). The integrated on-chip sonication followed by immunoprecipitation (IP) reaction can significantly reduce sample loss and contamination. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. We demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. Using our approach, we successfully detected the translocation of native NF-kappa B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. Droplets with encapsulated beads and cells have been increasingly used for studying molecular and cellular biology. However, a mixed population of droplets with an uneven number or type of encapsulated particles is resulted and used for screening. We developed a fluorescence-activated microfluidic droplet sorter that integrated a simple deflection mechanism. By passing droplets through a narrow interrogation channel, the encapsulated particles were detected individually. The microcontroller conducted the computation to determine the number and type of encapsulated particles in each droplet and made the sorting decision. Our results showed high efficiency and accuracy for sorting and enrichment. / Ph. D.

The shift of medical technology from a doctor's application of individualized medicine toward precision medicine has been accelerated by the advent of Next Generation Sequencing. Individualized medicine is where a doctor tries to understand the intricacies of a patient's medical state, where precision medicine uses a wealth of data to understand the individuality of a patient on a biological level to determine treatment course. Next Generation Sequencing allows for the collection of genome wide analyses such as genomic, transcriptomic, and epigenomic sequencing, which provides the backbone of the data driven precision medicine. In order to obtain and use this data, it needs to be produced from minimal amounts of patient tissue, such as the amount from a needle biopsy. In order to perform so many different assays it is paramount that we develop high sensitivity methodologies, such that we can gain an understanding of the patient's physiology without causing much discomfort in gathering large amounts of sample. In pursuit of making more tests, data, and assays available for use in precision medicine, we have developed 3 different microfluidic technologies, which automate and simplify the assays needed for the data collection at a high sensitivity, as well as a versatile platform for therapeutic production. First, we developed a epigenomic assay for chromatin immunoprecipitation, which gives us information on histone modifications across the genome. These histone modifications heavily impact gene expression and how the chromatin is organized, as well promoting or inhibiting transcription of genes. Our technology allowed us to perform multiple parallel assays from as few as 50 cells quickly and reliably using our fluidized bed technology. Next, we developed a library preparation system, which reduces the cost of library preparation by 20x and reduces operator pipetting by 100x. Our system uses a droplet based reactor to quickly and reliably prepare sequencing libraries using the lowest amount of DNA to date, 10 pg. Finally, we designed a therapeutics-on-a-chip platform which is capable of producing clinically relevant proteins on demand from temperature stable components. Using our system, we are capable of producing a number of different therapeutics on demand quickly without rearrangement of the system. / Doctor of Philosophy / Technical advances in the healthcare industry have made a range of new data available to physicians and patients. Home use DNA testing kits have made it possible to examine one’s predisposition to certain genetic diseases. Using these advanced methods, we are able to gain insights into a patient’s disease state where we were previously unable. Unfortunately, some of these new analyses currently require large amounts of patient sample, which make the examinations largely impractical to perform. In order to overcome the sample requirements, which make these analyses impractical, we develop microscale reactor systems capable of reducing the amount of material required for these new analyses. Here I demonstrate our developed technologies to automate 3 different processes aimed at enabling the study of protein-DNA interactions and produce therapeutics at the point of care. First, we developed an analytical system to study protein-DNA interactions (which are important to understanding patient responses to treatment), that allow for parallel analyses which can be done with sample from less than one needle biopsy, where existing methods would require dozens or more (50 vs 10,000,000 cells.) Next, we developed automated system for preparing DNA sequencing libraries using as little as 10 pg DNA (~2 cells of DNA). The device run multiple reactions simultaneously while reducing batch to batch variation and operator hands-on time. Finally, we developed a v Therapeutics-On-a-Chip platform that produces clinically relevant therapeutic proteins in clinically relevant dosages using a cell-free approach, while saving the trouble and cost associated with protein storage and transportation.

The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR. 327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines. These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease. / Master of Science

Since the dawn of the genomics era, the genetics of numerous human disorders has been understood which has led to improvements in targeted therapeutics. However, the focus of most research has been primarily on protein coding genes, which account for only 2% of the entire genome, leaving much of the remaining genome relatively unstudied. In particular, repetitive sequences, called microsatellites (MST), which are tandem repeats of 1 to 6 bases, are known to be mutational hotspots and have been linked to diseases, such as Huntington disease and Fragile X syndrome. This work represents a significant effort towards closing this knowledge gap. Specifically, we developed a next generation sequencing based enrichment method along with the supporting computational pipeline for detecting novel MST sequences in the human genome. Using this global MST enrichment protocol, we have identified 790 novel sequences. Analysis of these novel sequences has identified previously unknown functional elements, demonstrating its potential for aiding in the completion of the euchromatic DNA. We also developed a disease risk diagnostic using a novel target specific enrichment method that produces high resolution MST sequencing data that has the potential to validate, for the first time, the link between MST genotype variation and cancer. Combined with publicly available exome datasets of non-small cell lung cancer and 1000 genomes project, the target specific MST enrichment method uncovered a signature set of 21 MST loci that can differentiate between lung cancer and non-cancer control samples with a sensitivity ratio of 0.93. Finally, to understand the molecular causes of MST instability, we analyzed genomic variants and gene expression data for an autosomal recessive disorder, Fanconi anemia (FA). This first of its kind study quantified the heterogeneity of FA cells and demonstrated the possibility of utilizing the DNA crosslink repair dysfunctional FA cells as a suitable system to further study the causes of MST instability. / Ph. D.

Background: Prostate cancer (PCa) is one of the most prevalent cancers worldwide. The clinical manifestations and molecular characteristics of PCa are highly variable. Aggressive types require radical treatment, whereas indolent ones may be suitable for active surveillance or organ-preserving focal therapies. Patient stratification by clinical or pathological risk categories still lacks sufficient precision. Incorporating molecular biomarkers, such as transcriptome-wide expression signatures, improves patient stratification but so far excludes chromosomal rearrangements. In this study, we investigated gene fusions in PCa, characterized potential novel candidates, and explored their role as prognostic markers for PCa progression. Methods: We analyzed 630 patients in four cohorts with varying traits regarding sequencing protocols, sample conservation, and PCa risk group. The datasets included transcriptome-wide expression and matched clinical follow-up data to detect and characterize gene fusions in PCa. With the fusion calling software Arriba, we computationally predicted gene fusions. Following detection, we annotated the gene fusions using published databases for gene fusions in cancer. To relate the occurrence of gene fusions to Gleason Grading Groups and disease prognosis, we performed survival analyses using the Kaplan–Meier estimator, log-rank test, and Cox regression. Results: Our analyses identified two potential novel gene fusions, MBTTPS2,L0XNC01::SMS and AMACR::AMACR . These fusions were detected in all four studied cohorts, providing compelling evidence for the validity of these fusions and their relevance in PCa. We also found that the number of gene fusions detected in a patient sample was significantly associated with the time to biochemical recurrence in two of the four cohorts (log-rank test, p-value < 0.05 for both cohorts). This was also confirmed after adjusting the prognostic model for Gleason Grading Groups (Cox regression, p-values < 0.05). Conclusions: Our gene fusion characterization workflow revealed two potential novel fusions specific for PCa. We found evidence that the number of gene fusions was associated with the prognosis of PCa. However, as the quantitative correlations were only moderately strong, further validation and assessment of clinical value is required before potential application.

Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine. / Ph. D. / Microfluidics is a technology that manipulates solution with extremely small volume. It is an emerging platform that has revolutionized how molecular biology studies are conducted. It permits profiling of genome wide DNA changes or DNA-related changes (e.g. epigenomics) for a wide range of applications. One of the major contribution of microfluidics is to improve the next generation sequencing (NGS) technologies with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. DNA methylation and histone modification have been proved to affect a lot of biological processes, such as disease development. These developed technologies would benefit the development of precision medicine (a medical model that proposes the customization of healthcare) and treatment to various diseases. We applied these technologies to study the epigenomic differences between several cell types in the mouse brain.

Lymphoma is a common hematopoietic malignancy in canines. Current diagnostic techniques to diagnose lymphoma are often invasive and expensive. Additionally, tumor heterogeneity complicates the accurate classification and diagnosis of specific subtypes, hindering the development of targeted therapy and prognostic assessments. We propose a minimally invasive liquid biopsy technique involving blood collection to detect cell-free DNA from tumors using Next-generation sequencing. We hypothesize that identical tumor aberrations can be found in matching plasma and tumor DNA. Five dogs diagnosed with B-cell lymphoma through flow cytometry or PAAR were enrolled in the study. Samples collected included: (1) blood for plasma (cfDNA), (2) tumor tissue fine-needle aspirates (tumor DNA), and (3) buccal swabs (genomic DNA, germline control). Whole Genome Sequencing was performed using Illumina NovaSeq 6000, and the sequenced output was analyzed with bioinformatics tools to detect somatic variants in plasma and tumor samples. Our results revealed many shared somatic variants between matched cfDNA and tumor DNA samples, with 1.7-49% of tumor variants also found in corresponding plasma samples. Shared variants constituted only 0.5-9% of all plasma somatic variants. Specific B-cell lymphoma mutations were identified in cfDNA, including MYC, POT1, and TRAF3, alongside other cancer-related genes. Tumor samples showed mutations in genes associated with canine and human B-cell lymphoma. This study suggests that tumor-specific genomic mutations can be detected in plasma, supporting the potential of liquid biopsy as a less invasive diagnostic tool. However, cfDNA may not capture the full genetic heterogeneity of tumors due to low tumor-derived DNA content in limited plasma volumes. / Master of Science / Lymphoma is a type of blood cancer affecting white blood cells. Canine lymphoma is a common neoplasia, with an incidence rate of 20 to 100 cases per 100,000 dogs, making it a significant research focus. Current diagnostic methods are invasive and costly. Additionally, the wide variety of tumor types in lymphoma makes it challenging to determine the exact subtypes, which is crucial for selecting the best treatment approach. To overcome these challenges, we proposed a less invasive method known as "liquid biopsy". This technique involves taking a blood sample of a dog to find cell-free DNA from tumor cells using Next-Generation Sequencing technologies. We aimed to see if blood DNA could provide the same information as tumor DNA. In our study, we worked with five dogs diagnosed with B-cell lymphoma through traditional methods. We collected blood, tissue from needle biopsies, and buccal swabs from each dog. We then performed DNA extraction and sequencing on these samples. Our findings showed that 1.7-50% of the mutations in tumor DNA were also detected in matched blood DNA, though these represented only a small fraction of all changes found in blood samples. Additionally, the blood samples also revealed mutations related to canine B-cell lymphoma in genes like MYC, POT1, and TRAF3. In conclusion, our study supports the use of liquid biopsy as a feasible and less invasive method to diagnose lymphoma in dogs. However, they might not show all genetic variations of the tumor due to limited tumor DNA content.