CAIM: Coverage-based Analysis for Identification of Microbiome.

Accurate taxonomic profiling of microbial taxa in a metagenomic sample is vital to gain insights into microbial ecology. Recent advancements in sequencing technologies have contributed tremendously toward understanding these microbes at species resolution through a whole shotgun metagenomic (WMS) approach. In this study, we developed a new bioinformatics tool, CAIM, for accurate taxonomic classification and quantification within both long- and short-read metagenomic samples using an alignment-based method. CAIM depends on two different containment techniques to identify species in metagenomic samples using their genome coverage information to filter out false positives rather than the traditional approach of relative abundance. In addition, we propose a nucleotide-count based abundance estimation, which yield lesser root mean square error than the traditional read-count approach. We evaluated the performance of CAIM on 28 metagenomic mock communities and 2 synthetic datasets by comparing it with other top-performing tools. CAIM maintained a consitently good performance across datasets in identifying microbial taxa and in estimating relative abundances than other tools. CAIM was then applied to a real dataset sequenced on both Nanopore (with and without amplification) and Illumina sequencing platforms and found high similality of taxonomic profiles between the sequencing platforms. Lastly, CAIM was applied to fecal shotgun metagenomic datasets of 232 colorectal cancer patients and 229 controls obtained from 4 different countries and primary 44 liver cancer patients and 76 controls. The predictive performance of models using the genome-coverage cutoff was better than those using the relative-abundance cutoffs in discriminating colorectal cancer and primary liver cancer patients from healthy controls with a highly confident species markers.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

A NOTCH3-CXCL12-driven myeloma-tumor niche signaling axis promotes chemoresistance in multiple myeloma.

Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to Bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells.

Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Reduced osteoprotegerin expression by osteocytes may contribute to rebound resorption after denosumab discontinuation.

Denosumab is an anti-RANKL Ab that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss, but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast numbers in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term, but not short-term, denosumab administration reduced osteoprotegerin (OPG) mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA, suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.

Growth and dormancy control of myeloma cells by mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) may have contrasting impacts on the progression of multiple myeloma (MM). Priming normal MSCs, by culturing them with MM cells, mimics the MSC-induced MM growth. We studied the contrasting effects of conditioned medium (CM) from unprimed or primed MSCs on growth of MM cells from newly diagnosed cases. We elucidated potential molecular pathways using global gene expression profiling and focused on the role of the mTOR2 component, RICTOR, as a novel mediator of dormancy in MM. Primed MSCs CM consistently increased proportions of proliferating cells and supported MM growth in 3-day (n = 20) and 10-day (n = 12) cultures, effects that were partially mediated through the IGF1 axis. In contrast, unprimed MSCs CM inhibited growth of MM cells in cases mainly from stages I/II MM. The genes most overexpressed in MM cells treated with primed MSCs CM were associated with cell cycle, DNA-damage repair, and proliferation; genes most overexpressed in MM cells treated with unprimed MSCs CM were associated with dormancy pathways including RICTOR (mTOR2 pathway), CXCR4, and BCL2. RICTOR protein level was induced by unprimed MSCs CM and was lower in KI67+ proliferating MM cells treated with primed MSCs CM. RICTOR was underexpressed in clinical relapse samples compared with baseline samples of the same patients. Inhibiting RICTOR expression in primary MM cells promoted their growth, and enforced expression of RICTOR in MM cell lines inhibited their growth. Our findings suggest that, after prolonged interactions with MM cells, bone marrow MSCs shift from MM-repressive to MM-permissive. AVAILABILITY OF DATA AND MATERIALS: Our institutional GEP data of MM cells from newly diagnosed patients used to show RICTOR expression have been deposited at Gene Expression Omnibus (GEO: GSE2658, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2658).

CReSIL: accurate identification of extrachromosomal circular DNA from long-read sequences.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is found in many eukaryotic species and cell types, including cancer, where eccDNAs with oncogenes drive tumorigenesis. Most studies of eccDNA employ short-read sequencing for their identification. However, short-read sequencing cannot resolve the complexity of genomic repeats, which can lead to missing eccDNA products. Long-read sequencing technologies provide an alternative to constructing complete eccDNA maps. We present a software suite, Construction-based Rolling-circle-amplification for eccDNA Sequence Identification and Location (CReSIL), to identify and characterize eccDNA from long-read sequences. CReSIL's performance in identifying eccDNA, with a minimum F1 score of 0.98, is superior to the other bioinformatic tools based on simulated data. CReSIL provides many useful features for genomic annotation, which can be used to infer eccDNA function and Circos visualization for eccDNA architecture investigation. We demonstrated CReSIL's capability in several long-read sequencing datasets, including datasets enriched for eccDNA and whole genome datasets from cells containing large eccDNA products. In conclusion, the CReSIL suite software is a versatile tool for investigating complex and simple eccDNA in eukaryotic cells.

Piezo1 stimulates mitochondrial function via cAMP signaling.

Mechanical signals stimulate mitochondrial function but the molecular mechanisms are not clear. Here, we show that the mechanically sensitive ion channel Piezo1 plays a critical role in mitochondrial adaptation to mechanical stimulation. The activation of Piezo1 induced mitochondrial calcium uptake and oxidative phosphorylation (OXPHOS). In contrast, loss of Piezo1 reduced the mitochondrial oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production in calvarial cells and these changes were associated with increased expression of the phosphodiesterases Pde4a and lower cyclic AMP (cAMP) levels. In addition, Piezo1 increased cAMP production and the activation of a cAMP-responsive transcriptional reporter. Consistent with this, cAMP was sufficient to increase mitochondrial OCR and the inhibition of phosphodiesterases augmented the increase in OCR induced by Piezo1. Moreover, the inhibition of cAMP production or activity of protein kinase A, a kinase activated by cAMP, prevented the increase in OCR induced by Piezo1. These results demonstrate that cAMP signaling contributes to the increase in mitochondrial OXPHOS induced by activation of Piezo1.

Transcriptomic Analyses Reveal Long Non-Coding RNA in Peripheral Blood Mononuclear Cells as a Novel Biomarker for Diagnosis and Prognosis of Hepatocellular Carcinoma.

Novel biomarkers are highly required for the diagnosis and predicting prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the profiles of long non-coding RNAs (lncRNAs) obtained from the peripheral blood mononuclear cells (PBMCs) of patients with HCC and PBMCs from a co-culture model using transcriptomic analysis. The differentially expressed lncRNAs (DElncRNAs) were then characterized and integrated as cancer-induced lncRNAs. Among them, three up-regulating DElncRNAs including MIR4435-2HG, SNHG9 and lnc-LCP2-1 and one down-regulating, lnc-POLD3-2, were identified. The functional analysis showed that these enriched lncRNAs were mainly associated with carcinogenesis and immune responses. Following further validation in PBMCs samples (100 HBV-related HCC, 100 chronic hepatitis B and 100 healthy controls), MIR4435-2HG, lnc-POLD3-2 and their combination were revealed to be sensitive biomarkers in discriminating HCC from non-HCC (AUROC = 0.78, 0.80, and 0.87, respectively), particularly among individuals with normal serum alpha-fetoprotein levels. Additionally, high circulating SNHG9 expression was shown to be an independent prognostic factor of overall survival in patients with HCC. These results indicate that determining these lncRNAs in PBMCs could serve as novel diagnostic and prognostic biomarkers for HBV-related HCC.

CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation.

Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.

Mmp13 deletion in mesenchymal cells increases bone mass and may attenuate the cortical bone loss caused by estrogen deficiency.

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal cells, but functional evidence for the mediators of these effects has only recently begun to emerge. We report that the matrix metalloproteinase 13 (MMP13) is the highest up-regulated gene in mesenchymal cells from mice lacking the estrogen receptor alpha (ERα). In sham-operated female mice with conditional Mmp13 deletion in Prrx1 expressing cells (Mmp13ΔPrrx1), the femur and tibia length was lower as compared to control littermates (Mmp13f./f). Additionally, in the sham-operated female Mmp13ΔPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were higher and osteoclast number at the endocortical surfaces was lower, whereas bone formation rate was unaffected. Notably, the decrease of cortical thickness caused by ovariectomy (OVX) in the femur and tibia of Mmp13f./f mice was attenuated in the Mmp13ΔPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell-derived MMP13 may regulate osteoclast number and/or activity, bone resorption, and bone mass. And increased production of mesenchymal cell-derived factors may be important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone.

Notch3 signaling between myeloma cells and osteocytes in the tumor niche promotes tumor growth and bone destruction.

In multiple myeloma (MM), communication via Notch signaling in the tumor niche stimulates tumor progression and bone destruction. We previously showed that osteocytes activate Notch, increase Notch3 expression, and stimulate proliferation in MM cells. We show here that Notch3 inhibition in MM cells reduced MM proliferation, decreased Rankl expression, and abrogated the ability of MM cells to promote osteoclastogenesis. Further, Notch3 inhibition in MM cells partially prevented the Notch activation and increased proliferation induced by osteocytes, demonstrating that Notch3 mediates MM-osteocyte communication. Consistently, pro-proliferative and pro-osteoclastogenic pathways were upregulated in CD138+ cells from newly diagnosed MM patients with high vs. low NOTCH3 expression. These results show that NOTCH3 signaling in MM cells stimulates proliferation and increases their osteoclastogenic potential. In contrast, Notch2 inhibition did not alter MM cell proliferation or communication with osteocytes. Lastly, mice injected with Notch3 knock-down MM cells had a 50% decrease in tumor burden and a 50% reduction in osteolytic lesions than mice bearing control MM cells. Together, these findings identify Notch3 as a mediator of cell communication among MM cells and between MM cells and osteocytes in the MM tumor niche and warrant future studies to exploit Notch3 as a therapeutic target to treat MM.

Current and Future Methodology for Quantitation and Site-Specific Mapping the Location of DNA Adducts.

Formation of DNA adducts is a key event for a genotoxic mode of action, and their presence is often used as a surrogate for mutation and increased cancer risk. Interest in DNA adducts are twofold: first, to demonstrate exposure, and second, to link DNA adduct location to subsequent mutations or altered gene regulation. Methods have been established to quantitate DNA adducts with high chemical specificity and to visualize the location of DNA adducts, and elegant bio-analytical methods have been devised utilizing enzymes, various chemistries, and molecular biology methods. Traditionally, these highly specific methods cannot be combined, and the results are incomparable. Initially developed for single-molecule DNA sequencing, nanopore-type technologies are expected to enable simultaneous quantitation and location of DNA adducts across the genome. Herein, we briefly summarize the current methodologies for state-of-the-art quantitation of DNA adduct levels and mapping of DNA adducts and describe novel single-molecule DNA sequencing technologies to achieve both measures. Emerging technologies are expected to soon provide a comprehensive picture of the exposome and identify gene regions susceptible to DNA adduct formation.

Cell-Free DNA Analysis by Whole-Exome Sequencing for Hepatocellular Carcinoma: A Pilot Study in Thailand.

Cell-free DNA (cfDNA) has been used as a non-invasive biomarker for detecting cancer-specific mutations. However, the mutational profile of cfDNA in Thai patients with hepatocellular carcinoma (HCC) has not been investigated. Here, we demonstrated the utility of using whole-exome sequencing (WES) of cfDNA to define the somatic mutation profiles of HCC in Thai patients. The comprehensive profile of cfDNA was determined with WES to identify variants in matched cfDNA and germline DNA from 30 HCC patients in Thailand who underwent nonoperative therapies. The level of cfDNA was higher in HCC patients compared with chronic hepatitis patients (p-value < 0.001). Single nucleotide variants were present in somatic genes in cfDNA, including in ZNF814 (27%), HRNR (20%), ZNF492 (20%), ADAMTS12 (17%), FLG (17%), OBSCN (17%), TP53 (17%), and TTN (17%). These same mutations were matched to HCC mutation data from The Cancer Genome Atlas (TCGA) and a previous Thai HCC study. The co-occurrence of HRNR and TTN mutations in cfDNA was associated with shorter overall survival in HCC patients (hazard ratio = 1.60, p-value = 0.0196). These findings indicate that the mutational profile of cfDNA accurately reflected that of HCC tissue and suggest that cfDNA could serve as a useful biomarker for diagnosis and prognosis in Thai HCC patients. In addition, we demonstrated the use of the pocket-sized sequencer of Oxford Nanopore Technology to detect copy-number variants in HCC tissues that could be applied for onsite clinical detection/monitoring of HCC.

Identification of BHLHE40 expression in peripheral blood mononuclear cells as a novel biomarker for diagnosis and prognosis of hepatocellular carcinoma.

Novel and sensitive biomarkers is highly required for early detection and predicting prognosis of hepatocellular carcinoma (HCC). Here, we investigated transcription profiles from peripheral blood mononuclear cells (PBMCs) of 8 patients with HCC and PBMCs from co-culture model with HCC using RNA-Sequencing. These transcription profiles were cross compared with published microarray datasets of PBMCs in HCC to identify differentially expressed genes (DEGs). A total of commonly identified of 24 DEGs among these data were proposed as cancer-induced genes in PBMCs, including 18 upregulated and 6 downregulated DEGs. The KEGG pathway showed that these enriched genes were mainly associated with immune responses. Five up-regulated candidate genes including BHLHE40, AREG, SOCS1, CCL5, and DDIT4 were selected and further validated in PBMCs of 100 patients with HBV-related HCC, 100 patients with chronic HBV infection and 100 healthy controls. Based on ROC analysis, BHLHE40 and DDIT4 displayed better diagnostic performance than alpha-fetoprotein (AFP) in discriminating HCC from controls. Additionally, BHLHE40 and DDIT4 had high sensitivity for detecting AFP-negative and early-stage HCC. BHLHE40 was also emerged as an independent prognostic factor of overall survival of HCC. Together, our study indicated that BHLHE40 in PBMCs could be a promising diagnostic and prognostic biomarker for HBV-related HCC.

Detection and Discrimination of DNA Adducts Differing in Size, Regiochemistry, and Functional Group by Nanopore Sequencing.

Chemically induced DNA adducts can lead to mutations and cancer. Unfortunately, because common analytical methods (e.g., liquid chromatography-mass spectrometry) require adducts to be digested or liberated from DNA before quantification, information about their positions within the DNA sequence is lost. Advances in nanopore sequencing technologies allow individual DNA molecules to be analyzed at single-nucleobase resolution, enabling us to study the dynamic of epigenetic modifications and exposure-induced DNA adducts in their native forms on the DNA strand. We applied and evaluated the commercially available Oxford Nanopore Technology (ONT) sequencing platform for site-specific detection of DNA adducts and for distinguishing individual alkylated DNA adducts. Using ONT and the publicly available ELIGOS software, we analyzed a library of 15 plasmids containing site-specifically inserted O6- or N2-alkyl-2'-deoxyguanosine lesions differing in sizes and regiochemistries. Positions of DNA adducts were correctly located, and individual DNA adducts were clearly distinguished from each other.

Estrogens decrease osteoclast number by attenuating mitochondria oxidative phosphorylation and ATP production in early osteoclast precursors.

Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17ß-estradiol (E2) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax-two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E2. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases "oxidative phosphorylation" and the expression of mitochondria complex I genes. Additionally, E2 decreased the activity of complex I and oxygen consumption rate. Similar to E2, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.

A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma.

Molecular biomarkers provide both diagnostic and prognostic results for patients with diffuse glioma, the most common primary brain tumor in adults. Here, we used a long-read nanopore-based sequencing technique to simultaneously assess IDH mutation status and MGMT methylation level in 4 human cell lines and 8 fresh human brain tumor biopsies. Currently, these biomarkers are assayed separately, and results can take days to weeks. We demonstrated the use of nanopore Cas9-targeted sequencing (nCATS) to identify IDH1 and IDH2 mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation of MGMT methylation levels not only at the promoter region, as with currently used methods, but also at CpGs across the proximal promoter region, the entirety of exon 1, and a portion of intron 1. We compared the methylation levels of all CpGs to MGMT expression in all cell lines and tumors and observed a positive correlation between intron 1 methylation and MGMT expression. Finally, we identified single nucleotide variants in 3 target loci. This pilot study demonstrates the feasibility of using nCATS as a clinical tool for cancer precision medicine.