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Single-cell Hi-C (scHi-C) is a collection of protocols for studying genomic interactions within individual cells. Although data analysis for scHi-C resembles data analysis for bulk Hi-C, the unique challenges of scHi-C, such as high noise and protocol-specific biases, require specialized data processing strategies. In this tutorial chapter, we focus on using pairtools, a suite of tools optimized for scHi-C data, demonstrating its application on a Drosophila snHi-C dataset. While centered on pairtools for snHi-C data, the principles outlined are applicable across scHi-C variants with minor adjustments. This educational chapter aims to guide researchers in using open-source tools for scHi-C analysis, emphasizing critical steps of contact pair extraction, detection of ligation junctions, filtration, and deduplication.

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New scientific techniques and methods are always needed to link the perpetrators to the incident or the crime scene. Recent microbiota studies based on NGS (Next-generation sequencing) show that various biological samples from crime scenes have the potential to be used in forensic investigations. Especially when DNA traces belonging to more than one person are insufficient to fully determine the genetic profile, a secret sample, such as a microbiota sample created by the suspect's touch, can be used. In this preliminary study, a fictionalized experimental model was designed to investigate the transfer and persistence of the hand microbiome on the knife handle, which has a high potential to be used in criminal incidents, by metagenomic analysis methods. In addition, it was aimed to determine the transfer of specific bacterial species identified only to the person among the five participants onto the knife handle and their persistence over time. In the first stage of the research, samples were collected from the hands of 5 volunteer participants using the swap method, including their palms. Then, after each participant held a different knife, samples were collected from the knife handles via swabs from different angles of the knives at 4 and 24 h and analyzed by metagenomic methods. The findings of this preliminary study showed that the heatmap graphs generated after UniFrac distance analysis were not successful in establishing any similarity between the hand samples and the post-transfer knife handle samples. Nonetheless, it was observed that the transfer of bacterial species detected in the hand samples to knives differed according to the individuals and some bacterial species were transferred to the knife samples held by the participants. The number of bacterial species detected that are specific to each participant's hand sample was 302 in total, and it was determined that a total of 8.28 % of these bacterial species were transferred to the knife handle samples of the 4th hour and 6.95 % to the knife samples of the 24th hour. In the presented study, considering the transfer of some bacterial species in the hand microbiome, which are effective in the variation between individuals, onto the knife; It has been evaluated that some rare bacterial species can be important potential markers to associate the object with the perpetrator.

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BACKGROUND: The prognosis of breast cancer patients with visceral metastasis (VM) is significantly worse than that of patients without VM. We aimed to evaluate EZH2 (enhancer of zeste homolog 2) mutation as a biomarker associated with VM. METHODS: Data from forty-nine patients with metastatic breast cancer (MBC) pathologically confirmed at our hospital between March 2016 and September 2018 were collected. Metastatic tissue samples were obtained via ultrasound-guided needle biopsy, and paired peripheral blood samples were also collected. Tissue and blood samples were subjected to targeted next-generation sequencing via a 247-gene panel. Stably transfected MDA-MB-231 cells expressing wild-type EZH2 (EZH2WT) or a mutant form of EZH2 (EZH2K515R) were generated. Cell proliferation, colony formation ability, migration and invasion abilities and apoptosis were assessed using CCK-8 assays, plate colony formation assays, Transwell chamber assays and flow cytometry. RESULTS: The incidence of EZH2 mutations in the VM subgroup was greater than that in the non-VM subgroup in the entire cohort (n = 49, 42.3% vs. 13.0%, p = 0.024) and in the triple-negative breast cancer (TNBC) subgroup (n = 20, 50.0% vs. 10.0%, p = 0.05). Patients carrying EZH2 mutations had a significantly greater risk of developing VM than did those in the non-EZH2 mutation group in the entire cohort (HR 2.9) and in the TNBC subgroup (HR 6.45). Multivariate analysis revealed that EZH2 mutation was an independent prognostic factor for VM (HR 2.99, p = 0.009) in the entire cohort and in the TNBC subgroup (HR 10.1, p = 0.006). Data from cBioPortal also showed that patients with EZH2 mutations had a significantly greater risk of developing VM (HR 3.1), and the time to develop VM was significantly earlier in the EZH2 mutation group (31.5 months vs. 109.7 months, p = 0.008). Multivariate analysis revealed that EZH2 mutation (HR 2.73, p = 0.026) was an independent factor for VM after breast cancer surgery. There was no correlation between EZH2 mutations and BRCA1/2 mutations. Most of the patients (81.8%) in our cohort who developed VM carried the "c.1544A > G (p.K515R)" mutation. Compared with EZH2WT MDA-MB-231 cells, EZH2K515R MDA-MB-231 cells had greater colony formation rates (p < 0.01), greater migration and invasion rates (p < 0.001), and lower apoptosis rates (p < 0.01). The proportion of S + G2/M phase cells in the EZH2K515R group was significantly greater than that in the EZH2WT group. CONCLUSIONS: EZH2 mutation is associated with VM development in breast cancer patients. The EZH2K515R mutation leads to VM and a poor prognosis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells.

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Objective: To investigate the clinical utility of Next Generation Sequencing (NGS) in molecular typing of endometrial carcinoma and its combined screening for Lynch Syndrome (LS). Methods: 90 patients diagnosed with endometrial carcinoma (EC) and receiving treatment at the Third Affiliated Hospital of Zhengzhou University between March 2022 and December 2023 were included in this study. Molecular typing and germline evaluation for LS were conducted using NGS on the Illumina platform. A retrospective analysis was performed to examine the clinical pathological characteristics, molecular mutation spectrum, and LS screening outcomes among patients with four distinct molecular subtyping categories. Results: Among the 90 cases of EC, 11 cases (12.2%) of POLE mut type, 19 cases (21.1%) of MMRd type, 6 cases (6.7%) of p53 abn type, and 54 cases (60%) of NSMP type were detected, with detailed analysis of their respective molecular characteristics. LS screening identified 9 cases (10%) of pathogenic germline mutations in MMR genes, including 3 cases of MLH1 germline mutations, 2 cases of PMS2, 2 of MSH2, and 2 of MSH6. Of the 9 LS patients, 7 were MMRd type and 2 NSMP type, with 7 cases showing abnormal MMR protein expression. Additionally, 6 cases with germline variants of uncertain significance in MMR genes were detected, including 2 MLH1, 1 MSH6, 2 MSH6, 1 PMS2, and 1 EPCAM. Conclusion: NGS enables precise molecular typing of endometrial carcinoma through the identification of mutations in the POLE, TP53, and MMR genes. Conducting germline mutation testing for MMR genes in all patients with endometrial carcinoma can effectively prevent instances of overlooked LS diagnoses. Nevertheless, the extensive expenses associated with NGS necessitate additional validation and investigation before its clinical implementation can be fully endorsed.

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HLA-DPB1*04:02:24 differs from HLA-DPB1*04:02:01:01 by a single synonmous nucleotide substitution at position 639 G>A.

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HLA-C*08:291, a novel HLA class I allele detected by next-generation sequencing.

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HLA-DQA1*05:115 shows an alanine at position 59 not described previously.

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Induced pluripotent stem cells (iPSCs) have emerged as a revolutionary tool in cell therapies due to their ability to differentiate into various cell types, unlimited supply, and potential as off-the-shelf cell products. New advances in iPSC-derived immune cells have generated potent iNK and iT cells which showed robust killing of cancer cells in animal models and clinical trials. With the advent of advanced genome editing technologies that enable the development of highly engineered cells, here we outline 12 strategies to engineer iPSCs to overcome limitations and challenges of current cell-based immunotherapies, including safety switches, stealth edits, avoiding graft-versus-host disease (GvHD), targeting, reduced lymphodepletion, efficient differentiation, increased in vivo persistence, stemness, metabolic fitness, homing/trafficking, and overcoming suppressive tumor microenvironment and stromal cell barrier. With the development of advanced genome editing techniques, it is now possible to insert large DNA sequences into precise genomic locations without the need for DNA double strand breaks, enabling the potential for multiplexed knock out and insertion. These technological breakthroughs have made it possible to engineer complex cell therapy products at unprecedented speed and efficiency. The combination of iPSC derived iNK, iT and advanced gene editing techniques provides new opportunities and could lead to a new era for next generation of cell immunotherapies.

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In metastatic breast cancer (MBC), blood is a source of circulating tumor cells (CTCs). CTCs may serve as a ''real-time liquid biopsy" as they represent metastatic tumor genetics better than primary tumor. PIK3CA is one of the most important oncogenes in treatment-unresponsive breast cancers. The aim of this study was to detect PIK3CA mutations and hereditary cancer variants in CTCs from MBC patients. Forty-seven blood samples were obtained from 20 MBC patients from at least 1/3 consecutive time points. CTCs were quantified using the CellSearch system and isolated from 11/20 patients with ≥5/7.5 ml CTCs (14/47 blood samples) using the DEPArray system. DNA was extracted and amplified to perform Sanger sequencing on PIK3CA gene. Sequencing revealed a pathogenic PIK3CA mutation in 2/11 (18 %) cases. Subsequently, we evaluated a 26-target hereditary gene panel by Next Generation Sequencing and identified a concomitant pathogenic mutation in the TP53 gene in a patient with a PIK3CA mutation. No pathogenic germline variants were found. Our data support the conclusion that CTCs analysis may be used to identify mutations in patients to identify those more likely to metastasize.

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Hot springs have tremendous significance due to their divulging physiochemical features. In the recent past, metagenomics has emerged as a unique methodology to explore microbiota as well as new biocatalysts possessing advantageous biochemical properties from hot springs. In the present study, metagenomics has been employed for microbial diversity exploration and identification of genes involved in various metabolic pathways among two hot springs, Manikaran and Tatapani, located in Himachal Pradesh, India. Taxonomic analysis of both metagenomes revealed the dominance of the Proteobacteria phylum. Genomic signatures of other bacterial phyla such as Chloroflexi, Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, and Firmicutes were also found in significant abundance in both the metagenomes. The abundance of microorganisms belonging to genera, especially Nitrospira, Thauera, Meiothermus, Thiobacillus, Massilia, and Anaerolinea, was reported to be prevalent in the hot springs. A significant amount of metagenomic data remained taxonomically unclassified, which indeed emphasizes the scientific importance of these thermoaquatic niches. The functional potential analysis of both metagenomes revealed pathways related to carbohydrate metabolism, followed by amino acid metabolism, energy metabolism, genetic information processing, metabolism of cofactors and vitamins, membrane transporter, and signal transduction. Exploration of biomass-modifying biocatalysts enumerated the presence of glycoside hydrolases, glycosyl transferases, polysaccharide lyases, and carbohydrate esterases in the metagenomic data. Together, these findings offer an in-depth understanding of the microbial inhabitants in North-Western Himalayan hot springs and their underlying potential for various biotechnological and industrial applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01248-z.

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A case of Eimonosis orientalis was reported in a 52-year-old male farmer who presented with cough, phlegm, fever, headache, and nausea for more than 4 days. Haemophilic cells and fungal spores were identified in the bone marrow smear and confirmed as Aemon orientalis by culture. The same bacteria were also isolated from blood cultures.

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Accurate knowledge of the genome, virus and bacteria that have invaded our bodies is crucial for diagnosing many human diseases. The field of bioinformatics encompasses the complex computational methods required for this purpose. Metagenomics employs next-generation sequencing (NGS) technology to study and identify microbial communities in environmental samples. This technique allows for the measurement of the relative abundance of different microbes. Various tools are available for detecting bacterial species in sequenced metagenomic samples. In this study, we focus on well-known taxonomic classification tools such as MetaPhlAn4, Centrifuge, Kraken2, and Bracken, and evaluate their performance at the species level using synthetic and real datasets. The results indicate that MetaPhlAn4 exhibited high precision in identifying species in the simulated dataset, while Kraken2 had the best area under the precision-recall curve (AUPR) performance. Centrifuge, Kraken2, and Bracken showed accurate estimation of species abundances, unlike MetaPhlAn4, which had a higher L2 distance. In the real dataset analysis with samples from an inflammatory bowel disease (IBD) research, MetaPhlAn4, and Kraken2 had faster execution times, with differences in performance at family and species levels among the tools. Enterobacteriaceae and Pasteurellaceae were highlighted as the most abundant families by Centrifuge, Kraken2, and MetaPhlAn4, with variations in abundance among ulcerative colitis (UC), Crohn's disease (CD), and control non-IBD (CN) groups. Escherichia coli (E. coli) has the highest abundance among Enterobacteriaceae species in the CD and UC groups in comparison with the CN group. Bracken overestimated E. coli abundance, emphasizing result interpretation caution. The findings of this research can assist in selecting the appropriate short-read classifier, thereby aiding in the diagnosis of target diseases.

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Wastewater-based epidemiology (WBE) has been previously used as a tool for pathogen identification within communities. After the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) outbreak, in 2020, Daughton proposed the implementation of a wastewater surveillance strategy that could determine the incidence of COVID-19 (coronavirus disease 2019) nationally. Individuals in various stages of SARS-CoV-2 infection, including presymptomatic, asymptomatic and symptomatic patients, can be identified as carriers of the virus in their urine, saliva, stool and other bodily secretions. Studies using this method were conducted to monitor the prevalence of the virus in high-density populations, such as cities but also in smaller communities, such as schools and college campuses. The aim of this pilot study was to assess the feasibility and effectiveness of wastewater surveillance in Bucharest, Romania, and wastewater samples were collected weekly from seven locations between July and September 2023. RNA (ribonucleic acid) extraction, followed by dPCR (digital polymerase chain reaction) analysis, was performed to detect viral genetic material. Additionally, NGS (next generation sequencing) technology was used to identify the circulating variants within the wastewater of Bucharest, Romania. Preliminary results indicate the successful detection of SARS-CoV-2 RNA in wastewater, providing valuable insights into the circulation of the virus within the community.

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BACKGROUND: A new pathogen detection tool, metagenomic next-generation sequencing (mNGS), has been widely used for infection diagnosis, but the clinical and diagnostic value of mNGS in urinary tract infection (UTI) remains inconclusive. This systematic review with meta-analysis aimed to investigate the efficacy of mNGS in treating UTIs. METHODS: A comprehensive literature search was performed in PubMed, Web of Science, Embase, and the Cochrane Library, and eligible studies were selected based on the predetermined criteria. The quality of the included studies was assessed via the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool, and the certainty of evidence (CoE) was measured by the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) score. Then, the positive detection rate (PDR), pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve of the summary receiver operating characteristic curve (AUROC) was estimated in Review Manager, Stata, and MetaDisc. Subgroup analysis, meta-regression, and sensitivity analysis were performed to reveal the potential factors that influence internal heterogeneity. RESULTS: A total of 17 studies were selected for further analysis. The PDR of mNGS was markedly greater than that of culture (odds ratio (OR) = 2.87, 95% confidence interval [CI]: 1.72-4.81, p < 0.001, I2 = 90%). The GRADE score presented a very low CoE. Then, the pooled sensitivity was 0.89 (95% CI: 0.86-0.91, I2 = 39.65%, p = 0.06), and the pooled specificity was 0.75 (95% CI: 0.51-0.90, I2 = 88.64%, p < 0.001). The AUROC of the studies analyzed was 0.89 (95% CI: 0.86-0.92). The GRADE score indicated a low CoE. CONCLUSION: The current evidence shows that mNGS has favorable diagnostic performance for UTIs. More high-quality prospective randomized controlled trials (RCTs) are expected to verify these findings and provide more information about mNGS in UTI treatment and prognosis.

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Mycosis fungoides (MF), the predominant form of cutaneous T-cell lymphoma (CTCL), poses diagnostic challenges due to its clinical and histological resemblance to benign skin disorders. Delayed diagnosis contributes to therapeutic delays, prompting exploration of advanced diagnostics tools. Next-generation sequencing (NGS) may enhance disease detection by identifying pathogenic variants common to CTCL but absent in benign inflammatory disorders. We aim to discuss novel and common pathogenic variants in CTCL to enhance the utility of NGS as a diagnostic adjunct. This pilot study employed (NGS) to identify pathogenic variants in 10 MF cases. Cases were selected based on PCR-confirmed T-cell receptor clonality, with adequate DNA for NGS. GatorSeq NGS Panel, Illumina NextSeq500, and QIAGEN Clinical Insight QCI software facilitated sequencing, analysis, and variant interpretation. NGS revealed eight novel mutations in genes including HLA-DRB1, AK2, ITPKB, HLA-B, TYRO3, and CHD2. Additionally, previously reported MF-associated mutations such as DNMT3A, STAT5B, and SOCS1 (mouse study only) were detected as well. Detected variants were involved in apoptotic, NF-kB, JAK-STAT, and TCR signaling pathways, providing insights into MF pathogenesis. Mutations in genes like APC, AK2, TYRO3, and ITPKB that regulate tumor proliferation and apoptosis were noted. MF cases were associated with HLA gene mutations. NGS may enhance MF diagnosis, as the detection of pathogenic variants, particularly those known to occur in MF, favors a neoplastic diagnosis over an inflammatory diagnosis. Continuing this work may lead to the discovery of therapeutic targets.

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In April 2023, an outbreak of acute hepatitis was reported amongst internally displaced persons in the Nazareth community of South Sudan. IgM serology-based screening suggested the likely etiologic agent to be Hepatitis E virus (HEV). In this study, plasma specimens collected from anti-HEV IgM-positive cases were subjected to additional RT-qPCR testing and sequencing of extracted nucleic acids, resulting in the recovery of five full and eight partial HEV genomes. Maximum likelihood phylogenetic reconstruction confirmed the genomes belong to HEV genotype 1. Using distance-based methods, we show that genotype 1 is best split into three sub-genotypes instead of the previously proposed seven, and that these sub-genotypes are geographically restricted. The South Sudanese sequences confidently cluster within sub-genotype 1e, endemic to northeast, central, and east Africa. Bayesian Inference of phylogeny incorporating sampling dates shows that this new outbreak is not directly descended from other recent local outbreaks for which sequence data is available. However, the analysis suggests that sub-genotype 1e has been consistently and cryptically circulating locally for at least the past half century and that the known outbreaks are often not directly descended from one another. The ongoing presence of HEV, combined with poor sanitation and hygiene in the conflict-affected areas in the region, place vulnerable populations at risk for infection and its more serious effects, including progression to fulminant hepatitis.

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The draft genome data for Cimex hemipterus obtained through Illumina HiSeq sequencing were presented. The raw genomic data was deposited in GenBank under BioProject (PRJNA722579) with the BioSample accession number SAMN18780126. Software, including FLASH, SPADES, and QUAST, were used to merge, assemble, and qualify the raw dataset. The assembled genome was available in the Figshare repository. The assembled genomic data was compared to C. hemipterus data obtained using 454 Roche shotgun sequencing (BioProject, PRJNA308532), downloaded from NCBI. The draft genome data from this work demonstrated larger data volumes and an updated assembly of the C. hemipterus genome with better scaffolding compared to genome data obtained from 454 Roche shotgun sequencing.

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Introduction: Alzheimer's disease and related dementias (ADRD) represent a substantial global public health challenge with multifaceted impacts on individuals, families, and healthcare systems. Brief cognitive screening tools such as the Mini-Cog© can help improve recognition of ADRD in clinical practice, but widespread adoption continues to lag. We compared the Digital Clock and Recall (DCR), a next-generation process-driven adaptation of the Mini-Cog, with the original paper-and-pencil version in a well-characterized clinical trial sample. Methods: DCR was administered to 828 participants in the Bio-Hermes-001 clinical trial (age median ± SD = 72 ± 6.7, IQR = 11; 58% female) independently classified as cognitively unimpaired (n = 364) or as having mild cognitive impairment (MCI, n = 274) or dementia likely due to AD (DLAD, n = 190). MCI and DLAD cohorts were combined into a single impaired group for analysis. Two experienced neuropsychologists rated verbal recall accuracy and digitally drawn clocks using the original Mini-Cog scoring rules. Inter-rater reliability of Mini-Cog scores was computed for a subset of the data (n = 508) and concordance between Mini-Cog rule-based and DCR scoring was calculated. Results: Inter-rater reliability of Mini-Cog scoring was good to excellent, but Rater 2's scores were significantly higher than Rater 1's due to variation in clock scores (p < 0.0001). Mini-Cog and DCR scores were significantly correlated (τ B = 0.71, p < 0.0001). However, using a Mini-Cog cut score of 4, the DCR identified more cases of cognitive impairment (n = 47; χ 2 = 13.26, p < 0.0005) and Mini-Cog missed significantly more cases of cognitive impairment (n = 87). In addition, the DCR correctly classified significantly more cognitively impaired cases missed by the Mini-Cog (n = 44) than vice versa (n = 4; χ 2 = 21.69, p < 0.0001). Discussion: Our findings demonstrate higher sensitivity of the DCR, an automated, process-driven, and process-based digital adaptation of the Mini-Cog. Digital metrics capture clock drawing dynamics and increase detection of diagnosed cognitive impairment in a clinical trial cohort of older individuals.