An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies.

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.

Gut microbiota predicts severity and reveals novel metabolic signatures in acute pancreatitis.

OBJECTIVE: Early disease prediction is challenging in acute pancreatitis (AP). Here, we prospectively investigate whether the microbiome predicts severity of AP (Pancreatitis-Microbiome As Predictor of Severity; P-MAPS) early at hospital admission. DESIGN: Buccal and rectal microbial swabs were collected from 424 patients with AP within 72 hours of hospital admission in 15 European centres. All samples were sequenced by full-length 16S rRNA and metagenomic sequencing using Oxford Nanopore Technologies. Primary endpoint was the association of the orointestinal microbiome with the revised Atlanta classification (RAC). Secondary endpoints were mortality, length of hospital stay and severity (organ failure >48 hours and/or occurrence of pancreatic collections requiring intervention) as post hoc analysis. Multivariate analysis was conducted from normalised microbial and corresponding clinical data to build classifiers for predicting severity. For functional profiling, gene set enrichment analysis (GSEA) was performed and normalised enrichment scores calculated. RESULTS: After data processing, 411 buccal and 391 rectal samples were analysed. The intestinal microbiome significantly differed for the RAC (Bray-Curtis, p value=0.009), mortality (Bray-Curtis, p value 0.006), length of hospital stay (Bray-Curtis, p=0.009) and severity (Bray-Curtis, p value=0.008). A classifier for severity with 16 different species and systemic inflammatory response syndrome achieved an area under the receiving operating characteristic (AUROC) of 85%, a positive predictive value of 67% and a negative predictive value of 94% outperforming established severity scores. GSEA revealed functional pathway units suggesting elevated short-chain fatty acid (SCFA) production in severe AP. CONCLUSIONS: The orointestinal microbiome predicts clinical hallmark features of AP, and SCFAs may be used for future diagnostic and therapeutic concepts. TRIAL REGISTRATION NUMBER: NCT04777812.

The four seasons of pancreatitis - Etiology of acute pancreatitis during the course of the year.

BACKGROUND/OBJECTIVES: Whether seasonality is a factor that influences the incidence of acute pancreatitis (AP) is an under-investigated area. If seasonal incidence peaks can be detected, specifically with regard to biliary pancreatitis, has so far been answered in contradictory ways in the literature. METHODS: All AP cases from two tertiary German referral centers were identified between 2016 and 2022 based on ICD-10 discharge codes. The χ2 test for goodness of fit was applied to test significant differences in monthly and seasonal distributions of AP admissions. RESULTS: In total, 3597 AP cases were included. We observed significantly more idiopathic and biliary cases in May to July (p-values 0.041 and 0.027, respectively). Furthermore, most drug-induced APs were identified during the winter months (p-value 0.006). Moreover, there was a significant peak of alcohol-induced pancreatitis in summer and fall (p-value 0.038). CONCLUSIONS: Our data indicate a seasonal impact on AP incidences for certain etiologies.

Dynamics of intestinal and intratumoral microbiome signatures in genetically engineered mice and human pancreatic ductal adenocarcinoma.

BACKGROUND: Emerging evidence has recently revealed a prominent role of the microbiome in pancreatic ductal adenocarcinoma (PDAC). However, while most observations were made in patients, mouse models still require a precise characterization of their disease-related microbiome to employ them for mechanistic and interventional preclinical studies. METHODS: To investigate the fecal and tumoral microbiome of LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) and control (CTRL) mice, Oxford Nanopore sequencing was applied. Feces were collected from 10 KPC mice and 10 CTRLs at 3 timepoints (6 weeks, 12 weeks, and when tumor-bearing (KPC) or 6 months (CTRL), respectively). Metagenomic sequencing was performed on feces DNA. KPC tumor and healthy pancreas DNA samples were subjected to 16S rRNA gene sequencing. Bacterial marker components were detected in KPC tumor tissue over time by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). RESULTS: Murine fecal samples showed a significantly different microbiome compared to age-matched healthy CTRLs regarding beta diversity (p = 0.001, R2 = 0.2-0.25 for Bray-Curtis). Adjusted human PDAC classifiers predicted disease status from feces of KPC mice achieving area under the receiver operating characteristic (AUROC) values of 80%. Furthermore, KPC tumors harbored significantly more bacterial components than healthy pancreas. Also the microbial composition differs significantly between KPC tumors and healthy pancreas tissue (p = 0.042 for Bray-Curtis). Microbiota found highly abundant in human PDAC samples were considerably more abundant in KPC tumors as compared to healthy pancreas samples (p-value <0.001). CONCLUSION: KPC fecal samples show similarities with the microbial composition of stool samples from human PDAC patients.

[Metastasis or sarcoidosis - a diagnostic pitfall in metastatic rectal carcinoma]. / Metastase oder Sarkoidose ­ eine diagnostische Herausforderung beim metastasierten Rektumkarzinom.

BACKGROUND: Granulomatous diseases as sarcoidosis can impair the staging of metastatic diseases since metastasis are hard to distinguish from granulomas in standard imaging. This case report describes the diagnostic workup and therapy in a patient with simultaneous sarcoidosis and rectal cancer with hepatic metastasis and how a curative stadium was achieved. CASE DESCRIPTION: A 71-year old male patient was diagnosed with an adenocarcinoma of the rectum after presenting with involuntary weight loss and anemia. Further diagnostics raised strong suspicion of hepatic, pulmonary and splenic metastasis. Histology after bronchoscopy surprisingly discovered non-caseating granulomas, leading to the diagnosis of sarcoidosis. Due to an obstructive tumor, a rectum resection was performed. Due to a high suspicion of splenic metastasis during surgery, the spleen was removed. Histology revealed no metastasis in the spleen but multiple granulomas due to sarcoidosis. After surgery, biopsy of a suspicious lesion in the liver revealed both metastasis and sarcoidosis in the same sample. The patient was treated with a pseudo(neo)adjuvant chemotherapy with 5-Fluorouracil, Leukovorin, Oxaliplatin (FOLFOX) and Panitumumab (Anti-EGF-antibody). After treatment, CT scan revealed two hepatic lesions decreasing in size, while all other lesions were metrically stable. A right hemihepatectomy followed and histology revealed both sarcoidosis and metastasis. The curated patient was sent to aftercare and there is no suspicion for a relapse (13 month after last surgery). DISCUSSION: The simultaneous appearance of metastatic tumors and sarcoidosis creates a diagnostic dilemma since both manifestations can barely be distinguished in regular imaging technologies. This case report demonstrates that the histological work-up of affected organs with consecutive resections can cure a patient of a metastatic tumor disease, even in the context of simultaneous sarcoidosis.

[Acute necrotizing pancreatitis with hemorrhagic shock in secondary splenic rupture: a case report and literature review]. / Akute nekrotisierende Pankreatitis mit hämorrhagischem Schock bei sekundärer Milzruptur: Ein Fallbericht und Literaturübersicht.

Atraumatic splenic rupture is a rare complication of acute and chronic pancreatitis. It arises due to its anatomical proximity to the pancreas, for instance, due to erosion of large pseudocysts or walled-of-necrosis (WON).Following we describe the case of a 62-year-old woman who presented for further diagnostics and treatment of acute pancreatitis with the development of large walled-of necrosis (WON) in the pancreatic corpus and tail. During the course, the patient developed a hemorrhagic shock. An emergency computer tomography (CT) of the abdomen revealed a ruptured spleen with a large capsular hematoma with no evidence of active bleeding. In contrast to previous published case reports, our treatment was exclusively minimal-invasive: by radiological guided embolization of the splenic artery and by endosonographic guided implantation of a lumen apposing metal stent (LAMS). The splenic hematoma was spontaneously regressive without secondary drainage.

[Chronic pancreatitis: update diagnostic and therapeutic concepts]. / Chronische Pankreatitis: Update Diagnostik und Therapie.

INTRODUCTION: Chronic pancreatitis (CP) is a frequent cause for hospitalization and is associated with impaired quality of life and reduced overall survival. The German Society for Gastroenterology (DGVS) has recently completed the S3-Guideline "Pancreatitis" that summarizes key findings on epidemiology, diagnostic and therapeutic concepts for acute and chronic pancreatitis. Here, we recapitulate the most relevant findings for clinicians regarding CP. RESULTS: The most common cause of CP is chronic alcohol abuse, other causes are hereditary pancreatitis, autoimmune pancreatitis, hyperparathyroidism and idiopathic forms. Apart from the classical hereditary pancreatitis (PRSS1 mutation), a number of genetic associations have been discovered over the last years that are associated with an increased risk to develop idiopathic CP. The conservative management of CP is focused on the appropriate management of exocrine and endocrine insufficiency, and the prevention and treatment of secondary complications such as osteoporosis, vitamin deficiencies and malnutrition. Local complications (bile duct stenosis, duodenal stenosis, pseudocysts and chronic pain) should be managed in multidisciplinary teams in specialized pancreas centres with expert surgeons, radiologists and gastroenterologists. Infected or symptomatic pseudocysts should be primarily addressed by endoscopic drainage. In contrast, patients with chronic pain, dilated pancreas duct and opioid use should be considered for early surgical intervention. CONCLUSION: Chronic pancreatitis is associated with increased morbidity and mortality and often leads to hospital admissions. The clinical management of complex patients with local complications requires an interdisciplinary approach to tailor available therapeutic modalities depending on the stage of the disease and pre-existing comorbidities.

Management of Severe Acute Pancreatitis: An Update.

BACKGROUND: Severe acute pancreatitis (AP) continues to be a serious gastrointestinal disease with relevant morbidity and mortality. SUMMARY: Successful clinical management requires close interdisciplinary cooperation and coordination from experienced gastroenterologists, intensive care physicians, surgeons, and radiologists. While the early phase of the disease is characterized by intensive care aspects that focus primarily on treatment of organ failure, later complications are characterized especially by (infected) necrotic collections. Here, we discuss current clinical standards and developments for conservative and interventional management of patients with severe AP. Key messages: Early targeted fluid therapy within the first 48 h is critical to improve the outcome of severe AP. Thoracic epidural analgesia may have prognostically beneficial effects due to suspected anti-inflammatory effects and increased perfusion of splanchnic vessels. Enteral feeding should be started early during severe AP. Persistent organ failure (>48 h) is the strongest predictor of poor prognosis, and local complications such as infected walled-off necrosis should be primarily treated by minimally invasive endoscopic step-up approaches that are usually superior to surgical therapy options.

[Challenges in diagnostics for intestinal tuberculosis - Pitfalls of a forgotten infectious disease: case series and literature review]. / Herausforderungen in der Diagnostik der Darmtuberkulose ­ Pitfalls einer vergessenen infektiösen Erkrankung: Eine Fallserie und Literaturübersicht.

Intestinal tuberculosis is an infectious disease of the extrapulmonary manifestation with the Mycobacteria tuberculosis complex. In developed countries, this disease is rarely seen. The clinical features are heterogeneous and unspecific. Furthermore, intestinal tuberculosis poses diagnostic challenges. Regarding intestinal tuberculosis the Ziehl-Neelsen staining for acid-fast bacillus, PCR examination and culture methods show only poor sensitivity and specificity. In this case series, we present three patients suffering from intestinal tuberculosis, who were diagnosed and treated successfully. Furthermore, we review the literature about the pitfalls of the diagnostic approaches and the treatment options of intestinal tuberculosis.

Expression of gemcitabine metabolizing enzymes and stromal components reveal complexities of preclinical pancreatic cancer models for therapeutic testing.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) poorly responds to antineoplastic agents. Discrepancies between preclinical success and clinical failure of compounds has been a continuous challenge and major obstacle in PDAC research. AIM: To investigate the association of the tumor microenvironment (TME) composition and gemcitabine metabolizing enzyme (GME) expression in vitro and several in vivo models. METHODS: mRNA expression and protein levels of GME (cytosolic 5'-nucleotidase 1 A; NT5C1A, cytidine deaminase; CDA, deoxycytidine kinase; DCK), gemcitabine transporters (ENT1, ENT2, RRM1, RRM2) and stromal components (hyaluroninc acid, podoplanin, masson trichrome, picrosirius) were assessed by qRT-PCR and immunohistochemistry in murine LSL-KrasG12D/+;LSL-Trp53R172 H/+; Pdx-1-Cre (KPC), orthotopically transplanted mice (OTM), human primary resected PDAC tissue (hPRT), corresponding patient-derived xenograft (PDX) mice, and KPC-SPARC-/- mice. mRNA expression of GME was analyzed in PDAC cell lines (Panc-1, MIA PaCa, BXPC3 and L3.6) upon incubation on collagen or pancreatic stellate cell (PSC) conditioned media by qRT-PCR. RESULTS: Endogenous KPC tumors exhibited significantly higher levels of GME compared to OTM. However, GME levels did not differ between hPRT and corresponding PDX mice. Using Kendalls Tau correlation coefficient we did not show a significant correlation of GME and components of the TME except for NT5C1A and hyaluronic acid in PDX mice (p=0.029). GME were not significantly altered upon SPARC depletion in vivo, and upon treatment with PSC-conditioned media or incubation on collagen plated dishes in vitro. CONCLUSIONS: Our findings suggest that the expression of GME is independent from the deposition of stromal components. KPC mice are most appropriate to study stromal composition whereas PDX mice maintain GME expression of the corresponding hPRT and could be best suited for pharmacokinetic studies.

Machine learning-based decision tool for selecting patients with idiopathic acute pancreatitis for endosonography to exclude a biliary aetiology.

BACKGROUND: Biliary microlithiasis/sludge is detected in approximately 30% of patients with idiopathic acute pancreatitis (IAP). As recurrent biliary pancreatitis can be prevented, the underlying aetiology of IAP should be established. AIM: To develop a machine learning (ML) based decision tool for the use of endosonography (EUS) in pancreatitis patients to detect sludge and microlithiasis. METHODS: We retrospectively used routinely recorded clinical and laboratory parameters of 218 consecutive patients with confirmed AP admitted to our tertiary care hospital between 2015 and 2020. Patients who did not receive EUS as part of the diagnostic work-up and whose pancreatitis episode could be adequately explained by other causes than biliary sludge and microlithiasis were excluded. We trained supervised ML classifiers using H2O.ai automatically selecting the best suitable predictor model to predict microlithiasis/sludge. The predictor model was further validated in two independent retrospective cohorts from two tertiary care centers (117 patients). RESULTS: Twenty-eight categorized patients' variables recorded at admission were identified to compute the predictor model with an accuracy of 0.84 [95% confidence interval (CI): 0.791-0.9185], positive predictive value of 0.84, and negative predictive value of 0.80 in the identification cohort (218 patients). In the validation cohort, the robustness of the prediction model was confirmed with an accuracy of 0.76 (95%CI: 0.673-0.8347), positive predictive value of 0.76, and negative predictive value of 0.78 (117 patients). CONCLUSION: We present a robust and validated ML-based predictor model consisting of routinely recorded parameters at admission that can predict biliary sludge and microlithiasis as the cause of AP.

Time to Deliver on Promises: The Role of ERBB2 Alterations as Treatment Options for Colorectal Cancer Patients in the Era of Precision Oncology.

Receptor tyrosine kinase erythroblastic oncogene B2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2), represents an oncogenic driver and has been effectively targeted in breast and gastric cancer. Recently, next-generation sequencing (NGS) discovered ERBB2 as a promising therapeutic target in metastatic colorectal cancer (mCRC), where it is altered in 3-5% of patients, but no therapies are currently approved for this use. Herein, we present the experience of a single center in diagnosing actionable genetic ERBB2 alterations using NGS and utilizing the latest therapeutic options. Between October 2019 and December 2022, a total of 107 patients with advanced CRC underwent molecular analysis, revealing actionable ERBB2 mutations in two patients and ERBB2 amplifications in two other patients. These findings correlated with immunohistochemical (IHC) staining. Of these four patients, two were treated with trastuzumab-deruxtecan (T-DXd). We present two exemplary cases of patients with actionable ERBB2 alterations to demonstrate the effectiveness of T-DXd in heavily pretreated ERBB2-positive mCRC patients and the need for early molecular profiling. To fully exploit the potential of this promising treatment, earlier molecular profiling and the initiation of targeted therapies are essential.

The Microbiome in PDAC-Vantage Point for Future Therapies?

Microorganisms have been increasingly implicated in the pathogenesis of malignant diseases, potentially affecting different hallmarks of cancer. Despite the fact that we have recently gained tremendous insight into the existence and interaction of the microbiome with neoplastic cells, we are only beginning to understand and exploit this knowledge for the treatment of human malignancies. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor with limited therapeutic options and a poor long-term survival. Recent data have revealed fascinating insights into the role of the tumoral microbiome in PDAC, with profound implications for survival and potentially therapeutic outcomes. In this review, we outline the current scientific knowledge about the clinical and translational role of the microbiome in PDAC. We describe the microbial compositions in healthy and tumoral pancreatic tissue and point out four major aspects of the microbiome in PDAC: pathogenesis, diagnosis, treatment, and prognosis. However, caution must be drawn to inherent pitfalls in analyzing the intratumoral microbiome. Among others, contamination with environmental microbes is one of the major challenges. To this end, we discuss different decontamination approaches that are crucial for clinicians and scientists alike to foster applicability and physiological relevance in this translational field. Without a definition of an exact and reproducible intratumoral microbial composition, the exploitation of the microbiome as a diagnostic or therapeutic tool remains theoretical.

Activity of acute pancreatitis is modified by secreted protein acidic and rich in cysteine ablation.

BACKGROUND: Acute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood. OBJECTIVE: To investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein-induced AP in mice. METHODS: AP was induced by repeated cerulein injections in SPARC knock-out mice (SPARC-/- ) and control littermates (SPARC+/+ ). Secreted protein acidic and rich in cysteine expression and severity of AP were determined by histopathological scoring, immunohistochemistry, and biochemical assays. For functional analysis, primary murine acinar cell cultures with subsequent amylase release assays were employed. Proteome profiler assay and ELISA were conducted from pancreatic tissue lysates, and co-immunofluorescence was performed. RESULTS: Upon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC+/+ and SPARC-/- was indistinguishable. Notably, immune cell derived C-C Motif Chemokine Ligand 2 (CCL2) was highly elevated in SPARC+/+ pancreatic tissue potentially linking PSC derived SPARC with CCL2 induction in AP. CONCLUSION: SPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

[Diagnosis and Treatment of Chronic Pancreatitis]. / Chronische Pankreatitis und Autoimmunpankreatitis ­ Ein Update.

Chronic pancreatitis is a complex chronic inflammatory condition that results in pancreas fibrosis and calcification with subsequent exocrine and endocrine insufficiency. Alcohol and nicotine are the most prevalent risk factors, however, recent studies have increasingly discovered genetic associations that increase the risk to develop chronic pancreatitis. Here, we discuss different etiologies of the disease including autoimmune pancreatitis, and present evidence-based diagnostic and therapeutic approaches. In particular, we highlight the interdisciplinary and interventional management of local complications such as pseudocysts, pancreatic duct stones, biliary duct and pancreatic duct stenosis and persistent pain.

USP22 Suppresses SPARC Expression in Acute Colitis and Inflammation-Associated Colorectal Cancer.

As a member of the 11-gene "death-from-cancer" gene expression signature, ubiquitin-specific protease 22 (USP22) has been considered an oncogene in various human malignancies, including colorectal cancer (CRC). We recently identified an unexpected tumor-suppressive function of USP22 in CRC and detected intestinal inflammation after Usp22 deletion in mice. We aimed to investigate the function of USP22 in intestinal inflammation as well as inflammation-associated CRC. We evaluated the effects of a conditional, intestine-specific knockout of Usp22 during dextran sodium sulfate (DSS)-induced colitis and in a model for inflammation-associated CRC. Mice were analyzed phenotypically and histologically. Differentially regulated genes were identified in USP22-deficient human CRC cells and the occupancy of active histone markers was determined using chromatin immunoprecipitation. The knockout of Usp22 increased inflammation-associated symptoms after DSS treatment locally and systemically. In addition, Usp22 deletion resulted in increased inflammation-associated colorectal tumor growth. Mechanistically, USP22 depletion in human CRC cells induced a profound upregulation of secreted protein acidic and rich in cysteine (SPARC) by affecting H3K27ac and H2Bub1 occupancy on the SPARC gene. The induction of SPARC was confirmed in vivo in our intestinal Usp22-deficient mice. Together, our findings uncover that USP22 controls SPARC expression and inflammation intensity in colitis and CRC.

Comprehensive Wet-Bench and Bioinformatics Workflow for Complex Microbiota Using Oxford Nanopore Technologies.

The advent of high-throughput sequencing techniques has recently provided an astonishing insight into the composition and function of the human microbiome. Next-generation sequencing (NGS) has become the gold standard for advanced microbiome analysis; however, 3rd generation real-time sequencing, such as Oxford Nanopore Technologies (ONT), enables rapid sequencing from several kilobases to >2 Mb with high resolution. Despite the wide availability and the enormous potential for clinical and translational applications, ONT is poorly standardized in terms of sampling and storage conditions, DNA extraction, library creation, and bioinformatic classification. Here, we present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatic evaluation for complex microbiomes sequenced with ONT. Our findings from buccal and rectal swabs and DNA extraction experiments indicate that methods that were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Our benchmarking experiments reveal thresholds for analysis parameters that achieved excellent precision, recall, and area under the precision recall values and is superior to existing classifiers (Kraken2, Kaiju, and MetaMaps). Hence, our workflow provides an experimental and bioinformatic pipeline to perform a highly accurate analysis of complex microbial structures from buccal and rectal swabs. IMPORTANCE Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.