Nanopore Sequencing Data Analysis of 16S rRNA Genes Using the GenomeSync-GSTK System.

With the development of nanopore sequencing technology, long reads of DNA sequences can now be determined rapidly from various samples. This protocol introduces the GenomeSync-GSTK system for bacterial species identification in a given sample using nanopore sequencing data of 16S rRNA genes as an example. GenomeSync is a collection of genome sequences designed to provide easy access to genomic data of the species as demanded. GSTK (genome search toolkit) is a set of scripts for managing local homology searches using genomes obtained from the GenomeSync database. Based on this protocol, nanopore sequencing data analyses of metagenomes and amplicons could be efficiently performed. We also noted reanalysis in conjunction with future developments in nanopore sequencing technology and the accumulation of genome sequencing data.

Donor cord blood aging accelerates in recipients after transplantation.

Cord blood stem cell transplantation is an important alternative for patients needing hematopoietic stem cell transplantation. However, it is unclear how cord blood cells, which are 0 years old, age in the recipient's body after allogeneic transplantation. We performed DNA methylation (DNAm) age analysis to measure the age of cells using post-transplant peripheral blood in 50 cases of cord blood transplantation. The median chronological age (the time elapsed from the date of the cord blood transplant to the day the sample was taken for DNAm analysis) of donor cells was 4.0 years (0.2-15.0 years), while the median DNAm age was 10.0 years (1.3-30.3 years), and the ratio of DNAm age to chronological age (AgeAccel) was 2.7 (1.2-8.2). When comparing the mean values of AgeAccel in cord blood transplant cases and controls, the values were significantly higher in cord blood transplant cases. The characteristics of patients and transplant procedures were not associated with AgeAccel in this analysis, nor were they associated with the development of graft-versus-host disease. However, this analysis revealed that transplanting 0-year-old cord blood into a recipient resulted in cells aging more than twice as quickly as the elapsed time. The results shed light on the importance of the mismatch between cord blood stem cells and donor environmental factors in stem cell aging.

Robotic data acquisition with deep learning enables cell image-based prediction of transcriptomic phenotypes.

Single-cell whole-transcriptome analysis is the gold standard approach to identifying molecularly defined cell phenotypes. However, this approach cannot be used for dynamics measurements such as live-cell imaging. Here, we developed a multifunctional robot, the automated live imaging and cell picking system (ALPS) and used it to perform single-cell RNA sequencing for microscopically observed cells with multiple imaging modes. Using robotically obtained data that linked cell images and the whole transcriptome, we successfully predicted transcriptome-defined cell phenotypes in a noninvasive manner using cell image-based deep learning. This noninvasive approach opens a window to determine the live-cell whole transcriptome in real time. Moreover, this work, which is based on a data-driven approach, is a proof of concept for determining the transcriptome-defined phenotypes (i.e., not relying on specific genes) of any cell from cell images using a model trained on linked datasets.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

RELN rs7341475 Associates with Brain Structure in Japanese Healthy Females.

Although temperament has been regarded as an innate aspect of human personality, its association with proteins involved in embryonic development is unclear. Reelin, encoded by RELN, plays an important role in brain development. Herein, we investigated the association between the RELN rs7341475 (G/A) single nucleotide polymorphism, detected as a female-specific risk factor for schizophrenia, brain structure, and temperament to elucidate the role of RELN in the development of human personality. In this study, 1580 healthy young Japanese adults were genotyped for RELN rs7341475 and completed the Temperament and Character Inventory. Whole-brain analysis of covariance was conducted to investigate differences between genotypes in regional gray matter volume (rGMV) and cortical morphology. Additionally, multiple regression analysis was performed to examine the association of four temperaments with rGMV. Those statistical analyses were performed separately for males and females. Individuals with G/G homozygosity showed significantly greater rGMV in several areas of the brain, particularly the bilateral cingulate and temporal gyrus, as well as a larger value of fractal dimension in the left lateral occipital cortex. Furthermore, of the four temperaments, the novelty seeking was significantly and positively associated with rGMV in the right superior temporal gyrus, partially overlapping with areas where differences between the rs7341475 genotypes were detected. The above findings were detected only in females, but not in males. This is the first study to demonstrate the contribution of RELN rs7341475 to differences in brain structure in Japanese females, which may indicate vulnerability to schizophrenia and variations in human personality.

Regression tree analysis of the relationship between the concentrations of antimicrobial components and the microbiota of normal milk from dairy cows.

The purpose of this study was to determine the concentrations of antimicrobial components (immunoglobulin A (IgA), lactoferrin (LF), lingual antimicrobial peptide (LAP), and S100A7) in normal milk and their relation to host factors (Age, somatic cell count (SCC), days in milk, richness, and alpha diversity of the milk microbiota) in dairy cows using multivariate regression tree analyses, and to clarify how the milk microbiota is related to the obtained results. Thirty normal milk samples were collected from a commercial dairy farm in June 2020. The thresholds that predicted the concentration of each antimicrobial component in milk were obtained by regression tree analysis, and the beta-diversity of the milk microbiota composition between groups divided according to each threshold was compared by an analysis of similarities test. The IgA and LF concentrations were mainly predicted by the SCC (177,500 and 70,000 cells/ml, respectively), and the LAP and S100A7 concentrations were predicted by Age (29.667 and 40.3 months, respectively). No relationship was observed between the concentration of IgA, LAP, or S100A7 and the milk microbiota composition between the groups divided by the threshold for prediction, but the milk microbiota composition was significantly different between the groups divided by the threshold for predicting the LF concentration. Our results indicated that the LF concentration in normal milk may be associated with the milk microbiota composition.

Examination of the microbiota of normal cow milk using MinIONTM nanopore sequencing.

The aim of this study was to evaluate the microbiota of normal milk in dairy cows and their relationship with host factors, such as the age of the cow (Age), somatic cell counts in milk (SCCs), and days in milk (DIM). We investigated 48 milk samples from 22 cows with no systemic or local clinical signs using MinIONTM nanopore sequencing for a 16S rRNA gene amplicon. Bacterial richness was positively correlated with the DIM (P=0.043), and both the Shannon-Wiener Index and Simpson's Index, which are metrics of alpha-diversity, were also significantly positively correlated with the SCC (P<0.001). The composition ratios of both Actinobacteria at the phylum level and Kocuria spp. at the genus level in the milk microbiota were significantly correlated with the SCC (P<0.001 and P<0.001, respectively). In the beta-diversity test, the one-way analysis of similarities test showed a significant difference (P=0.0051) between the low- and high-SCC groups. This study clarified that the composition of the normal milk microbiota in this herd was related to the SCC. It also raised the possibility of variations in bacterial genera in the normal milk microbiota between the low- and high-SCC groups. However, to clarify the actual condition of the milk microbiota and to elucidate the relationship with the SCC, it is necessary to perform further analyses taking into account not only the relative abundance, but also the absolute abundance of microbes.

Polygenic risk score for bipolar disorder associates with divergent thinking and brain structures in the prefrontal cortex.

It has been hypothesized that a higher genetic risk of bipolar disorder (BD) is associated with greater creativity. Given the clinical importance of bipolar disorder and the importance of creativity to human society and cultural development, it is essential to reveal their associations and the neural basis of the genetic risk of bipolar disorder to gain insight into its etiology. However, despite the previous demonstration of the associations of polygenic risk score (PRS) of BD and creative jobs, the associations of BD-PRS and creativity measured by the divergent thinking (CMDT) and regional gray matter volume (rGMV) as well as regional white matter volume (rWMV) have not been investigated. Using psychological analyses and whole-brain voxel-by-voxel analyses, we examined these potential associations in 1558 young, typically developing adult students. After adjusting for confounding variables and multiple comparisons, a greater BD-PRS was associated with a greater total CMDT fluency score, and a significant relationship was found in fluency subscores. A greater BD-PRS was also associated with lower total mood disturbance. Neuroimaging analyses revealed that the BD-PRS was associated with greater rGMV in the right inferior frontal gyrus, which is a consistently affected area in BD, as well as a greater rWMV in the left middle frontal gyrus, which has been suggested to play a central role in the increased creativity associated with the risk of BD with creativity. These findings suggest a relationship between the genetic risk of BD and CMDT and prefrontal cortical structures among young educated individuals.

Plasmid analysis of NDM metallo-ß-lactamase-producing Enterobacterales isolated in Vietnam.

Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148-149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75-76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

Occupational Hypersensitivity Pneumonitis in a Japanese Citrus Farmer.

Hypersensitivity pneumonitis (HP) sometimes develops in people working in specific environments. We herein report a case of occupation-related HP in a citrus farmer in Japan. A 66-year-old man developed a fever, dyspnea, and general malaise in March after working near a trash dump filled with moldy tangerines. He presented with leukocytosis, bilateral lung opacities on chest radiographs, and intra-alveolar and interstitial lymphocytic inflammation with fibrotic change on a lung biopsy. His symptoms disappeared after admission and recurred on a revisit to the workplace. Fungal culture and a mycobiome analysis using next-generation sequencing suggested an association with exposure to Penicillium digitatum.

Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing.

BACKGROUND: Although a microbiological diagnosis of pleural infection is clinically important, it is often complicated by prior antibiotic treatment and/or difficulties with culturing some bacterial species. Therefore, we aimed to identify probable causative bacteria in pleural empyema/parapneumonic effusions by combining 16S ribosomal RNA (rRNA) gene amplification and next-generation sequencing (NGS). METHODS: Pleural fluids were collected from 19 patients with infectious effusions and nine patients with non-infectious malignant effusions. We analysed DNA extracted from the pleural fluid supernatant by NGS using the Genome Search Toolkit and GenomeSync database, either directly or after PCR amplification of the 16S rRNA gene. Infectious and non-infectious effusions were distinguished by semi-quantitative PCR of the 16S rRNA gene. RESULTS: Only 8 (42%) effusions were culture-positive, however, NGS of the 16S rRNA gene amplicon identified 14 anaerobes and 7 aerobes/facultative anaerobes in all patients, including Streptococcus sp. (n = 6), Fusobacterium sp. (n = 5), Porphyromonas sp. (n = 5), and Prevotella sp. (n = 4), accounting for >10% of the total genomes. The culture and NGS results were discordant for 3 out of 8 patients, all of whom had previously been treated with antibiotics. Total (2ΔCT value in semi-quantitative PCR of the 16S rRNA gene) and specific (total bacterial load multiplied by the proportion of primary bacteria in NGS) bacterial loads could efficiently distinguish empyema/parapneumonic effusion from non-infectious effusion. CONCLUSION: Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria.

Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer.

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

Comprehensive genomic analysis reveals dynamic evolution of endogenous retroviruses that code for retroviral-like protein domains.

BACKGROUND: Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of mammalian germline cells. A large proportion of ERVs lose their open reading frames (ORFs), while others retain them and become exapted by the host species. However, it remains unclear what proportion of ERVs possess ORFs (ERV-ORFs), become transcribed, and serve as candidates for co-opted genes. RESULTS: We investigated characteristics of 176,401 ERV-ORFs containing retroviral-like protein domains (gag, pro, pol, and env) in 19 mammalian genomes. The fractions of ERVs possessing ORFs were overall small (~ 0.15%) although they varied depending on domain types as well as species. The observed divergence of ERV-ORF from their consensus sequences showed bimodal distributions, suggesting that a large proportion of ERV-ORFs either recently, or anciently, inserted themselves into mammalian genomes. Alternatively, very few ERVs lacking ORFs were found to exhibit similar divergence patterns. To identify candidates for ERV-derived genes, we estimated the ratio of non-synonymous to synonymous substitution rates (dN/dS) for ERV-ORFs in human and non-human mammalian pairs, and found that approximately 42% of the ERV-ORFs showed dN/dS < 1. Further, using functional genomics data including transcriptome sequencing, we determined that approximately 9.7% of these selected ERV-ORFs exhibited transcriptional potential. CONCLUSIONS: These results suggest that purifying selection operates on a certain portion of ERV-ORFs, some of which may correspond to uncharacterized functional genes hidden within mammalian genomes. Together, our analyses suggest that more ERV-ORFs may be co-opted in a host-species specific manner than we currently know, which are likely to have contributed to mammalian evolution and diversification.

Utility of Comprehensive Serum Glycopeptide Spectra Analysis (CSGSA) for the Detection of Early Stage Epithelial Ovarian Cancer.

Comprehensive serum glycopeptide spectra analysis (CSGSA) evaluates >10,000 serum glycopeptides and identifies unique glycopeptide peaks and patterns via supervised orthogonal partial least-squares discriminant modeling. CSGSA was more accurate than cancer antigen 125 (CA125) or human epididymis protein 4 (HE4) for detecting early stage epithelial ovarian cancer. Combined CSGSA, CA125, and HE4 had improved diagnostic performance. Thus, CSGSA may be a useful screening tool for detecting early stage epithelial ovarian cancer.

Comprehensive Serum Glycopeptide Spectra Analysis Combined with Artificial Intelligence (CSGSA-AI) to Diagnose Early-Stage Ovarian Cancer.

Ovarian cancer is a leading cause of deaths among gynecological cancers, and a method to detect early-stage epithelial ovarian cancer (EOC) is urgently needed. We aimed to develop an artificial intelligence (AI)-based comprehensive serum glycopeptide spectra analysis (CSGSA-AI) method in combination with convolutional neural network (CNN) to detect aberrant glycans in serum samples of patients with EOC. We converted serum glycopeptide expression patterns into two-dimensional (2D) barcodes to let CNN learn and distinguish between EOC and non-EOC. CNN was trained using 60% samples and validated using 40% samples. We observed that principal component analysis-based alignment of glycopeptides to generate 2D barcodes significantly increased the diagnostic accuracy (88%) of the method. When CNN was trained with 2D barcodes colored on the basis of serum levels of CA125 and HE4, a diagnostic accuracy of 95% was achieved. We believe that this simple and low-cost method will increase the detection of EOC.

Sequence Compression Benchmark (SCB) database-A comprehensive evaluation of reference-free compressors for FASTA-formatted sequences.

BACKGROUND: Nearly all molecular sequence databases currently use gzip for data compression. Ongoing rapid accumulation of stored data calls for a more efficient compression tool. Although numerous compressors exist, both specialized and general-purpose, choosing one of them was difficult because no comprehensive analysis of their comparative advantages for sequence compression was available. FINDINGS: We systematically benchmarked 430 settings of 48 compressors (including 29 specialized sequence compressors and 19 general-purpose compressors) on representative FASTA-formatted datasets of DNA, RNA, and protein sequences. Each compressor was evaluated on 17 performance measures, including compression strength, as well as time and memory required for compression and decompression. We used 27 test datasets including individual genomes of various sizes, DNA and RNA datasets, and standard protein datasets. We summarized the results as the Sequence Compression Benchmark database (SCB database, http://kirr.dyndns.org/sequence-compression-benchmark/), which allows custom visualizations to be built for selected subsets of benchmark results. CONCLUSION: We found that modern compressors offer a large improvement in compactness and speed compared to gzip. Our benchmark allows compressors and their settings to be compared using a variety of performance measures, offering the opportunity to select the optimal compressor on the basis of the data type and usage scenario specific to a particular application.

Implications of HLA diversity among regions for bone marrow donor searches in Japan.

Japan is an island country, and the Japanese people have had minimal genetic exchange with other ethnolinguistic groups. Consequently, the population is highly uniform and has limited HLA diversity relative to people from other countries. However, Japan has three ethnolinguistic groups, and HLA distributions differ depending on geographic region. To collect an HLA-rich variety of bone marrow bank donor registrants, it is essential to know the precise distribution of HLA in Japan. We analyzed HLA alleles and haplotypes based on HLA information of 177 041 bone marrow donor registrants. Registrants were grouped depending on the prefecture and region (a group of prefectures) as commonly used in Japan. The prefectures did not show the same distributions, but the tendency was similar for each region. We found that Okinawa Prefecture and the mainland can be clearly divided as haplotypes: [A*24:02-C*01:02-B*54:01-DRB1*04:05] and [A*24:02-C*01:02-B*59:01-DRB1*04:05] were typically found in Okinawa (P = .02, P < .001). Moreover, these types were found almost exclusively in Japan and Korea. Donor registration centers of the Japan Marrow Donor Program are currently located in all prefectures. It is essential to deploy registration centers to collect registrants with a large variety of HLA types covering all of Japan.

Usefulness of next-generation DNA sequencing for the diagnosis of urinary tract infection.

Acute urinary tract infection (UTI) is a highly common clinical condition. Although bacterial culture is the gold standard diagnostic test, false negative results may be possible, leading to the pathogen being unidentified. In recent years, bacterial DNA sequencing analysis has garnered much attention, but clinical studies are rare in Japan. In this study, we assessed the usefulness of next-generation DNA sequencing (NGS) analysis for acute UTI patients. We thus performed an observational, retrospective case series study. Urine and blood samples were collected from ten acute UTI patients, of whom four had also been diagnosed with urosepsis. Seven variable regions of bacterial 16S rRNA genes were amplified by PCR and then sequenced by IonPGM. The identified bacterial species were compared with those identified using the culture tests and the clinical parameters were analyzed. As a result, the NGS method effectively identified predominant culture-positive bacteria in urine samples. The urine NGS also detected several culture-negative species, which have been reported to be potentially pathogenic. Out of four urosepsis cases, three were pathogen-positive in blood NGS results, while two were pathogen-negative in blood culture. In one sepsis case, although blood culture was negative for Escherichia coli, this species was detected by blood NGS. For non-sepsis cases, however, blood NGS, as well as blood culture, was less effective in detecting bacterial signals. In conclusion, NGS is potentially useful for identifying pathogenic bacteria in urine from acute UTI patients but is less applicable in patients who do not meet clinical criteria for sepsis.

Molecular characterization of feline paramyxovirus in Japanese cat populations.

Feline paramyxovirus (FPaV) is a member of the family Paramyxoviridae that has been reported only in Germany and the United Kingdom. We detected FPaV for the first time in Japan by transcriptome sequencing of cat urine samples. We determined the genome structure of FPaV and conducted a phylogenetic analysis. It was found that FPaV belongs to the genus Jeilongvirus and forms a clade with Mount Mabu Lophuromys virus 1 (MMLV-1). FPaV lacks a small hydrophobic (SH) gene that is found in members of the genus Jeilongvirus; however, some jeilongviruses also do not have this gene. These results provide information about the diversity and evolution of paramyxoviruses.