High-throughput Sequencing Data Research Articles

Multiple approaches in the study of ancient human microbiome

Abstract Research related to the analysis of microbial communities in ancient human samples began two decades ago. Since then, new technologies as well as new bioinformatic tools have been developed for the manipulation of massive data. All this together, with the theoretical knowledge generated from microbial ecology has expanded our understanding of the human microbiome. Despite the challenge of studying ancient samples and their microbiome, some approaches applied to the contemporary human microbiome have been successfully applied to ancient samples. Most studies in ancient samples have focused on taxonomic profiling at the community level with metagenomic or high-throughput 16S rRNA gene amplicon sequencing data. However, other approaches have emerged in order to get deeper understanding of the ancient human microbiome. Therefore, the aim of this review is to discuss all the approaches and methodologies successfully applied to ancient samples and ancient microbiome as well as the information that can be generated using them, in order to understand the evolution of human microbiome interactions throughout history.

Microbial community structures and bacteria-Cylindrospermopsis raciborskii interactions in Yilong Lake.

Cylindrospermopsis raciborskii-dominated harmful algae blooms have been reported globally in recent years. However, our understanding of the ecology of C. raciborskii in natural conditions is still poor. In this study, we collected the water samples from a C. raciborskii-blooming lake, Yilong Lake, in Yunnan province, China, and used both culture-dependent and culture-independent approaches to investigate their microbial communities and the interactions between C. raciborskii and the other bacteria. The composition and diversity of microbial communities were revealed with 16S rRNA gene high-throughput sequencing data analysis. Microbial co-occurrences analysis suggests C. raciborskii may have complex associations with other bacteria. Based on co-inoculation tests, we obtained 14 strains of bacterial strains from the water samples that exhibited either algicidal or promoting effects on a strain of C. raciborskii. Two bacterial isolates exhibited a consistent performance between co-occurrence analysis and experimental results. Effects of these bacteria-algae interspecies interactions on the bloom event are discussed. All these results may provide new insights into the C. raciborskii-dominated blooms and how its interspecies relationships with other bacteria may influence the bloom events in eutrophic waters throughout the world.

Transcriptome Analysis of Tomato Leaves Reveals Candidate Genes Responsive to Tomato Brown Rugose Fruit Virus Infection.

Tomato brown rugose fruit virus (ToBRFV) is a newly-emerging tobamovirus which was first reported on tomatoes in Israel and Jordan, and which has now spread rapidly in Asia, Europe, North America, and Africa. ToBRFV can overcome the resistance to other tobamoviruses conferred by tomato Tm-1, Tm-2, and Tm-22 genes, and it has seriously affected global crop production. The rapid and comprehensive transcription reprogramming of host plant cells is the key to resisting virus attack, but there have been no studies of the transcriptome changes induced by ToBRFV in tomatoes. Here, we made a comparative transcriptome analysis between tomato leaves infected with ToBRFV for 21 days and those mock-inoculated as controls. A total of 522 differentially expressed genes were identified after ToBRFV infection, of which 270 were up-regulated and 252 were down-regulated. Functional analysis showed that DEGs were involved in biological processes such as response to wounding, response to stress, protein folding, and defense response. Ten DEGs were selected and verified by qRT-PCR, confirming the reliability of the high-throughput sequencing data. These results provide candidate genes or signal pathways for the response of tomato leaves to ToBRFV infection.

Enricherator: A Bayesian Method for Inferring Regularized Genome-wide Enrichments from Sequencing Count Data

A pervasive question in biological research studying gene regulation, chromatin structure, or genomics is where, and to what extent, does a signal of interest arise genome-wide? This question is addressed using a variety of methods relying on high-throughput sequencing data as their final output, including ChIP-seq for protein-DNA interactions,1 GapR-seq for measuring supercoiling,2 and HBD-seq or DRIP-seq for R-loop positioning.3,4 Current computational methods to calculate genome-wide enrichment of the signal of interest usually do not properly handle the count-based nature of sequencing data, they often do not make use of the local correlation structure of sequencing data, and they do not apply any regularization of enrichment estimates. This can result in unrealistic estimates of the true underlying biological enrichment of interest, unrealistically low estimates of confidence in point estimates of enrichment (or no estimates of confidence at all), unrealistic gyrations in enrichment estimates at very close (<10 bp) genomic loci due to noise inherent in sequencing data, and in a multiple-hypothesis testing problem during interpretation of genome-wide enrichment estimates. We developed a tool called Enricherator to infer genome-wide enrichments from sequencing count data. Enricherator uses the variational Bayes algorithm to fit a generalized linear model to sequencing count data and to sample from the approximate posterior distribution of enrichment estimates (https://github.com/jwschroeder3/enricherator). Enrichments inferred by Enricherator more precisely identify known binding sites in cases where low coverage between binding sites leads to false-positive peak calls in these noisy regions of the genome; these benefits extend to published datasets.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Occurrence of grapevine yellow speckle viroid 2 and Australian grapevine viroid in Idaho grapevines.

Grapevine yellow speckle viroid 2 (GYSVd-2; Pospiviroidae, Apscaviroid) causes yellow speckle disease in grapevine (Koltunow et al. 1989) and was found in Australia, Iran, Italy, China, and Nigeria (Koltunow et al. 1989; Habili 2017; Zongoma et al. 2018). In the U.S., GYSVd-2 was found in the State of Washington (Vitis vinifera L. cv. Merlot; Alabi et al., 2012). Australian grapevine viroid (AGVd; Pospiviroidae, Apscaviroid) was reported in Australia, Italy, China, Tunisia, Iran, and in the U.S. wine grapes (V. vinifera) (Habili 2017). In the U.S., AGVd was reported from California (Al Rwahnih et al. 2009), from Washington State (V. vinifera cv. Syrah; GU327604), and from the State of New York (an unknown cv. of V. vinifera; KY081960). In Idaho, two other viroids, hop stunt viroid (HSVd; Pospiviroidae, Hostuviroid) and grapevine yellow speckle viroid 1 (GYSVd-1; Pospiviroidae, Apscaviroid), common in grapevines were previously found in wine grapes (Thompson et al. 2019) but neither GYSVd-2 nor AGVd were identified in the same high-throughput sequencing (HTS) outputs. In September 2020, 16 leaf and petiole samples were collected from six vineyards in Canyon and Nez Perce counties of Idaho, representing six different wine grape cultivars and an unknown table grape cultivar, and subjected to HTS analysis. One of the samples was from a table grape plant at the edge of a declining 'Chardonnay' wine grape block that was grown next to a wine tasting room deck for aesthetic, ornamental purposes; the table grape and 'Chardonnay' plants were own-rooted and planted in 1981. Ribodepleted total RNAs prepared from these samples, as described previously, were subjected to a HTS analysis on a NovaSeq platform (Dahan et al. 2023), producing 15,095,042 to 31,500,611 250-bp paired-end reads per sample. Raw reads were adapter and quality cleaned and mapped against the V. vinifera, reference genome. Unmapped paired-end reads were assembled, and contigs were analyzed using BLASTn and DIAMOND (Buchfink et al. 2021) programs. Fifteen samples were found infected with HSVd and with GYSVd-1, while one was infected with GYSVd-2 and AGVd; in particular, the table grape plant (arbitrarily designated RBTG) was found infected with all four viroid species. The HTS-derived, 490-nt GYSVd-2-specific contig from the table grape sample represented ∼1.35 genome of the Idaho isolate of GYSVd-2 (GYSVd-2-RBTG) and was 100% identical to the GYSVd-2 sequence JQ686716 from Iran. The HTS-derived, 488-nt AGVd-specific contig represented ∼1.32 genome of the Idaho isolate of AGVd (AGVd-RBTG) and was 100% identical to the AGVd sequence KF876037 from Iran. To validate the HTS data and confirm the presence of the four viroids in the original 16 samples, all of them were subjected to RT-PCR using the viroid-specific primers described by Gambino et al. (2014); all 16 samples were found positive for HSVd and GYSVd-1, and one found positive for AGVd. The RBTG sample was confirmed to be infected with HSVd, GYSVd-1, and AGVd by RT-PCR. GYSVd-2 sequence was not amplified, although primers designed by Gambino et al. (2014) matched the HTS-derived GYSVd-2-RBTG sequence; this may be related to a lower concentration of this viroid in the sample and to properties of the primers. The sampled table grape plant was asymptomatic; all four viroids were apparently not associated with any visible abnormalities in this table grape plant, consistent with the findings that viroids found in grapevines typically do not seem to be associated with visible diseases (Habili 2017).

Distribution of Plasmopara viticola Causing Downy Mildew in Russian Far East Grapevines

Downy mildew is a severe disease that leads to significant losses in grape yields worldwide. It is caused by the oomycete Plasmopara viticola. The study of the distribution of this agent and the search for endophytic organisms that inhibit the growth of P. viticola are essential objectives to facilitate the transition to sustainable and high-yield agriculture, while respecting the environment. In this study, high-throughput sequencing of the ITS (ITS1f/ITS2 region) and 16S (V4 region) amplicons was employed to analyze 80 samples of leaves and stems from different grapevine species and cultivars grown in the Russian Far East (Vitis amurensis Rupr., Vitis coignetiae Pulliat, and several grapevine cultivars). The analysis revealed the presence of P. viticola in 53.75% of the grape samples. The pathogen P. viticola was not detected in V. amurensis samples collected near Vladivostok and Russky Island. Among the P. viticola-affected samples, only two (out of the eighty analyzed grape samples) from the Makarevich vineyard in Primorsky Krai exhibited disease symptoms, while the majority appeared visually healthy. We also found six distinct P. viticola ASVs in our metagenomic data. Based on phylogenetic analysis, we hypothesize that the P. viticola population in the Russian Far East may have originated from the invasive P. viticola clade aestivalis, which has spread around the world from North America. To identify putative microbial antagonists of P. viticola, a differential analysis of high-throughput sequencing data was conducted using the DESeq2 method to compare healthy and P. viticola-affected samples. The in silico analysis revealed an increased representation of certain taxa in healthy samples compared to P. viticola-affected ones: fungi—Kabatina sp., Aureobasidium sp., and Vishniacozyma sp.; bacteria—Hymenobacter spp., Sphingomonas spp., Massilia spp., Methylobacterium-Methylorubrum spp., and Chryseobacterium spp. This in-silico-obtained information on the potential microbial antagonists of P. viticola serves as a theoretical basis for the development of biocontrol agents for grapevine downy mildew.

Abstract 3484: Mechanism of BRAF V600E resistance revealed by cell panel screening and bioinformatics

Abstract The BRAF V600E mutation is pivotal target in the treatment of melanoma. Dabrafenib, one of the most potent inhibitors approved by the FDA, exhibits differential sensitivity across different cancer types. To explore the underlying mechanisms responsible for the resistance of certain cancers to Dabrafenib, we tested a panel of 20 BRAF V600E positive cancer cell lines. Our results showed that 15 of these cell lines demonstrated sensitivity to Dabrafenib, whereas the remaining 5 exhibited resistance. We then investigated the correlation between Dabrafenib potency and gene expression patterns based on public high throughput sequencing data via bioinformatics methods. The results uncovered that the cell lines resistant to Dabrafenib showed low expression of MAP3K1, alongside with elevated expression of G0S2 and PIK3CG. These alterations are known to promote tumor cell proliferation, migration, and invasion, thereby contributing to a reduced sensitivity to BRAF inhibitors. Subsequent in vitro experiments validated the findings derived from our analysis. This unbiased, bioinformatics-driven approach provides valuable insight into the reasons behind treatment resistance and aids in the development of next-generation treatment approaches for inhibitor-resistant cancers. Citation Format: Kaiqiang Hu, Qiuyuan Yang, Pengwei Pan, Fang He. Mechanism of BRAF V600E resistance revealed by cell panel screening and bioinformatics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3484.

Unraveling the role of microRNA-like RNAs in enhancing Puccinia triticina pathogenicity in wheat by high-throughput sequencing data

Wheat (Triticum aestivum L.), a staple crop cultivated across an estimated 215 million hectares worldwide, encounters significant yield reductions due to the leaf rust disease, instigated by the fungus Puccinia triticina (Pt). Control strategies for this pathogen are limited. Research on wheat-leaf rust interaction in the past few years has mainly focused on characterizing and modulating the performance of resistance (R) and other defense genes of the host plant, but rarely targeted to decipher the network of molecular strategies employed by the pathogen except secretome analysis. In wheat plants, pathogen may induce a greater diversity of small RNAs (sRNAs) to manipulate the host's gene expression and suppress defense responses. In the present study, Pt microRNA-like-RNAs (milRNAs) were screened and identified from sRNA libraries prepared from leaf rust pathogen-infected leaves of wheat near isogenic lines (NILs). Pathogenesis engendering Pt target genes were validated through degradome libraries. Our study indicates that milRNAs enter the host system to suppress its immunity and promote symbiosis-like association to maintain a longer virulence span. Through RT‒qPCR expression analysis, it was suggested that milRNAs create an environment to enhance symbiosis via cross-kingdom silencing of host disease resistance gene encoding proteins. We found that seven out of eight milRNAs effectively regulate pathogenesis-related genes and repressed the disease-resistance genes in host wheat. Functional annotation of target genes of Pt reveals regulation of vesicle synthesis, transport, and fusion, which represents cross-kingdom vesicle-mediated export of milRNAs. Our results also manifest an integrative view of how virulence-associated milRNAs endeavor to stamp out PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) to increase susceptibility of the host. This study epitomizes one of the tactics developed by Pt employing milRNAs in regulating virulence during leaf rust infection and will usher to design strategies to manage pathogenesis to protect wheat immunity against leaf rust disease.

Diversity, Community Structure, and Antagonism of Endophytic Fungi from Asymptomatic and Symptomatic Mongolian Pine Trees.

Diplodia tip blight, caused by Diplodia sapinea (=Sphaeropsis sapinea), are widely distributed in Honghuaerji, Inner Mongolia, China, causing severe damage on natural Mongolian pine (Pinus sylvestris var. mongolica). D. sapinea is an endophyte that becomes pathogenic under conditions of drought, hail damage, or temperature-associated stress. The role of the endophytic community inhabiting different pine tissues in the expression of disease is still unknown. In this study, the diversity and community structure of endophytic fungi among asymptomatic and symptomatic Mongolian pine were detected using culture-based isolation and high-throughput sequencing (HTS), and the potential antagonistic endophytes against D. sapinea were also screened. The results indicated that 198 and 235 strains of endophytic fungi were isolated from different tissues of symptomatic and asymptomatic Mongolian pine, respectively. D. sapinea was the most common endophyte isolated from the current-year needles and shoots of symptomatic trees, and Diplodia was also the most common in the HTS data. There were no significant differences in the endophytic fungal species richness among asymptomatic and symptomatic trees, but there were differences observed within specific sampled tissues. The ANOSIM analysis confirmed that the endophytic fungi community structure significantly differed between sampling tissues among symptomatic and asymptomatic Mongolian pine. Furthermore, the antagonism study revealed Penicillium fructuariae-cellae with the ability to inhibit the growth of D. sapinea in vitro, and the potential performance of this fungus, acting as biological control agent, was evaluated under greenhouse. Our findings can pave the way to a better understanding of the interactions between D. sapinea, other endophytic fungi and their hosts, and provide helpful information for more efficient disease management strategies.

Multi-omics reveals that alkaline mineral water improves the respiratory health and growth performance of transported calves

BackgroundLong-distance transportation, a frequent practice in the cattle industry, stresses calves and results in morbidity, mortality, and growth suppression, leading to welfare concerns and economic losses. Alkaline mineral water (AMW) is an electrolyte additive containing multiple mineral elements and shows stress-mitigating effects on humans and bovines.ResultsHere, we monitored the respiratory health status and growth performance of 60 Simmental calves subjected to 30 hours of road transportation using a clinical scoring system. Within the three days of commingling before the transportation and 30 days after the transportation, calves in the AMW group (n = 30) were supplied with AMW, while calves in the Control group (n = 29) were not. On three specific days, namely the day before transportation (day -3), the 30th day (day 30), and the 60th day (day 60) after transportation, sets of venous blood, serum, and nasopharyngeal swab samples were collected from 20 calves (10 from each group) for routine blood testing, whole blood transcriptomic sequencing, serology detection, serum untargeted metabolic sequencing, and 16S rRNA gene sequencing. The field data showed that calves in the AMW group displayed lower rectal temperatures (38.967 ℃ vs. 39.022 ℃; p = 0.004), respiratory scores (0.079 vs. 0.144; p < 0.001), appetite scores (0.024 vs. 0.055; p < 0.001), ocular and ear scores (0.185 vs. 0.338; p < 0.001), nasal discharge scores (0.143 vs. 0.241; p < 0.001), and higher body weight gains (30.870 kg vs. 7.552 kg; p < 0.001). The outcomes of laboratory and high throughput sequencing data revealed that the calves in the AMW group demonstrated higher cellular and humoral immunities, antioxidant capacities, lower inflammatory levels, and intestinal absorption and lipogenesis on days -3 and 60. The nasopharynx 16S rRNA gene microbiome analysis revealed the different composition and structure of the nasopharyngeal microflora in the two groups of calves on day 30. Joint analysis of multi-omics revealed that on days -3 and 30, bile secretion was a shared pathway enriched by differentially expressed genes and metabolites, and there were strong correlations between the differentially expressed metabolites and the main genera in the nasopharynx.ConclusionsThese results suggest that AMW supplementation enhances peripheral immunity, nutrition absorption, and metabolic processes, subsequently affecting the nasopharyngeal microbiota and improving the respiratory health and growth performance of transported calves. This investigation provided a practical approach to mitigate transportation stress and explored its underlying mechanisms, which are beneficial for the development of the livestock industry.9KT6u9sWWs8ZH5U7yZx1qCVideo

Elucidating the molecular mechanisms of sepsis: Identifying key aging-related biomarkers and potential therapeutic targets in the treatment of sepsis.

Sepsis remains a crucial global health issue characterized by high mortality rates and a lack of specific treatments. This study aimed to elucidate the molecular mechanisms underlying sepsis and to identify potential therapeutic targets and compounds. High-throughput sequencing data from the GEO database (GSE26440 as the training set and GSE13904 and GSE32707 as the validation sets), weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, alongside a combination of PPI and machine learning methods (LASSO and SVM) were utilized. WGCNA identified the black module as positively correlated, and the green module as negatively correlated with sepsis. Further intersections of these module genes with age-related genes yielded 57 sepsis-related genes. GO and KEGG pathway enrichment analysis, PPI, LASSO, and SVM selected six hub aging-related genes: BCL6, FOS, ETS1, ETS2, MAPK14, and MYC. A diagnostic model was constructed based on these six core genes, presenting commendable performance in both the training and validation sets. Notably, ETS1 demonstrated significant differential expression between mild and severe sepsis, indicating its potential as a biomarker of severity. Furthermore, immune infiltration analysis of these six core genes revealed their correlation with most immune cells and immune-related pathways. Additionally, compounds were identified in the traditional Chinese medicine Danshen, which upon further analysis, revealed 354 potential target proteins. GO and KEGG enrichment analysis of these targets indicated a primary enrichment in inflammation and immune-related pathways. A Venn diagram intersects these target proteins, and our aforementioned six core genes yielded three common genes, suggesting the potential efficacy of Danshen in sepsis treatment through these genes. This study highlights the pivotal roles of age-related genes in the molecular mechanisms of sepsis, offers potential biomarkers, and identifies promising therapeutic compounds, laying a robust foundation for future studies on the treatment of sepsis.

Optimising high-throughput sequencing data analysis, from gene database selection to the analysis of compositional data: a case study on tropical soil nematodes

Optimising high-throughput sequencing data analysis, from gene database selection to the analysis of compositional data: a case study on tropical soil nematodes

Construction of relatedness matrices in autopolyploid populations using low-depth high-throughput sequencing data

Key messageAn improved estimator of genomic relatedness using low-depth high-throughput sequencing data for autopolyploids is developed. Its outputs strongly correlate with SNP array-based estimates and are available in the package GUSrelate.High-throughput sequencing (HTS) methods have reduced sequencing costs and resources compared to array-based tools, facilitating the investigation of many non-model polyploid species. One important quantity that can be computed from HTS data is the genetic relatedness between all individuals in a population. However, HTS data are often messy, with multiple sources of errors (i.e. sequencing errors or missing parental alleles) which, if not accounted for, can lead to bias in genomic relatedness estimates. We derive a new estimator for constructing a genomic relationship matrix (GRM) from HTS data for autopolyploid species that accounts for errors associated with low sequencing depths, implemented in the R package GUSrelate. Simulations revealed that GUSrelate performed similarly to existing GRM methods at high depth but reduced bias in self-relatedness estimates when the sequencing depth was low. Using a panel consisting of 351 tetraploid potato genotypes, we found that GUSrelate produced GRMs from genotyping-by-sequencing (GBS) data that were highly correlated with a GRM computed from SNP array data, and less biased than existing methods when benchmarking against the array-based GRM estimates. GUSrelate provides researchers with a tool to reliably construct GRMs from low-depth HTS data.

KoNA: Korean Nucleotide Archive as A New Data Repository for Nucleotide Sequence Data

Abstract During the last decade, the generation and accumulation of petabase-scale high-throughput sequencing data have resulted in great challenges, including access to human data, as well as transfer, storage, and sharing of enormous amounts of data. To promote data-driven biological research, the Korean government announced that all biological data generated from government-funded research projects should be deposited at the Korea BioData Station (K-BDS), which consists of multiple databases for individual data types. We introduce the Korean Nucleotide Archive (KoNA), a repository of nucleotide sequence data. As of July 2022, the Korean Read Archive in KoNA has collected over 477 TB of raw next-generation sequencing data from national genome projects. To ensure data quality and prepare for international alignment, a standard operating procedure was adopted, which is similar to that of the International Nucleotide Sequence Database Collaboration. The standard operating procedure includes quality control processes for submitted data and metadata using an automated pipeline, followed by manual examination. To ensure fast and stable data transfer, a high-speed transmission system called GBox is used in KoNA. Furthermore, the data uploaded to or downloaded from KoNA through GBox can be readily processed using a cloud computing service called Bio-Express. This seamless coupling of KoNA, GBox, and Bio-Express enhances the data experience, including submission, access, and analysis of raw nucleotide sequences. KoNA not only satisfies the unmet needs for a national sequence repository in Korea but also provides datasets to researchers globally and contributes to advances in genomics. The KoNA is available at https://www.kobic.re.kr/kona/.

First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection.

Hibiscus is native to southeast Asia but well suited to Colombia's arid soil and dry climates from the coast to the mountains of Bogotá. Viruses infecting hibiscus in Colombia are largely unexplored, with four viruses previously known: hibiscus chlorotic ringspot virus (HCRSV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), and citrus leprosis virus C2 (CiLV-C2) (Padmanabhan et al., 2023). Mixed infections between these viruses were frequently detected. A recent virome analysis of a single hibiscus plant from Colombia revealed multiple viruses in mixed infection; : HCRSV, HLFPV, passion fruit green spot virus (PFGSV), a strain of physalis vein necrosis nepovirus, four novel carlavirus, one new potexvirus and a mitovirus. In addition, few smaller contigs of blunervirus and soymovirus were also identified in the high throughput sequencing (HTS) data, but their presence in the mixed infection could not be validated (A. Roy et al. 2023unpublish data). During Brevipalpus-transmitted virus (BTV) surveys, two asymptomatic and 15 hibiscus foliar samples showing green ringspots with central chlorotic spots in senescing areas, mosaic, and black or chlorotic spots were collected from six departments (states) in three geographical regions of Colombia: Tolima (n=4) and Cauca Valley (n=2) (Andean region), Meta (n=6) and Casanare (n=1) (Orinoquia region), and Quindío (n=1) and Risaralda (n=1) (coffee growing region). About 100 mg of 17 hibiscus leaf samples were separately processed for RNA isolation without DNase I treatment and tested for known BTVs, and for newly discovered hibiscus soymovirus (HSV; genus Soymovirus family Caulimoviridae) using PCR assays (Padmanabhan et al. 2023, Wang et al. 2023). To identify potential HSV infection in the samples, published SVF1/SVR1 and newly designed primer pairs (HSV-REP-F/-R and HSV-CPG-F/-R) were used to amplify the 430 nt transactivation (ORF-VI), 631 nt replicase (REP) and 401 nt coat protein gene (CPG), respectively (Supplementary 1). Of 17 samples tested, three from Tolima and one each from Meta and Quindío yielded all three expected size amplicons. Bi-directional sequencing followed by BLASTn analysis revealed 95-98% nt identity with the CPG, REP, and ORF-VI genes of HSV (OP757659). Ribo-depleted libraries were prepared using the RNA extracts of five HSV PCR positive samples. HTS yielded 11.6 to 50.3 million raw reads per sample library. Adapters were trimmed and filtered from the raw reads with Trimmomatic v0.39 and then assembled using SPAdes v3.15.5 (Padmanabhan et al., 2023). Contigs were blasted against the Arabidopsis proteome and a RefSeq-based viral protein database. Potential viral sequences were then blasted against the complete NCBI nr database. Assembled soymo contigs covered 99-100% of the HSV genome, with per-nucleotide read depths of 23.8 to 393. Contigs from the Tolima (Accessions; OR621030- OR621032 and Quindío samples (OR621033) covered 99-100% of the HSV genome and had >96-98% nt identity to Hawaiian isolate (OP757659) whereas the Meta sample contigs covered 78% of the genome with 9495% nt identity. HTS contigs shared >98-99% nt identities with their PCR amplicons. Along with HSV, other virus sequences (HCRSV, HLFPV, PFGSV, CiLV-C2, and mycoviruses) were variously detected from all five libraries. Due to mixed infection no symptom similarity was noticed among these 5 samples. The findings in hibiscus in Tolima, Meta and Quindío represent the first confirmed report of HSV infection in hibiscus in Colombia. The widespread distribution suggests the possibility of HSV dispersion via movement of planting material, and potential further spread to another hibiscus growing region.

PHDtools: A platform for pathogen detection and multi-dimensional genetic signatures decoding to realize pathogen genomics data analyses online

ObjectivesFacing the emerging diseases, rapid identification of the pathogen and multi-dimensional characterization of the genomic features at both isolate-level and population-level through high-throughput sequencing data can provide invaluable information to guide the development of antiviral agents and strategies. However, a user-friendly program is in urgent need for clinical laboratories without bioinformatics background to decode the complex big genomics data. MethodsIn this study, we developed an interactive online platform named PHDtools with a total of 15 functions to analyze metagenomics data to identify the potential pathogen and decode multi-dimensional genetic signatures including intra-/inter-host variations and lineage-level variations. The platform was applied to analyze the meta-genomic data of the samples collected from the 172 imported COVID-19 cases. ResultsAccording to the analytical results of mNGS, 27 patients were found to have the co-infections of SARS-CoV-2 with either influenza virus (n = 9) or human picobirnavirus (n = 19). Enough coverages of all the assembled SARS-CoV-2 genomes provided the sub-lineages of Omicron variant, and the number of mutations in the non-structural genes and M gene was increased, as well as the intra-host variations occurred in E and M gene were under positive selection (Ka/Ks > 1). These findings of increased or changed mutations in the SARS-CoV-2 genome characterized the current adaptive evolution patterns of Omicron sub-lineages, and revealed the evolution speed of these sub-lineages might increase. ConclusionsConsequently, the application of PHDtools has proved that this platform is accurate, user-friendly and convenient for clinical users who are deficient in bioinformatics, and the clinical monitor of SARS-CoV-2 genomes by PHDtools also highlighted the potential evolution features of current SARS-CoV-2 and indicated that the development of anti-SARS-CoV-2 agents and new-designed vaccines should incorporate the gene variations other than S gene.

Correlation between the bacterial flora and sensory quality of Bayannur sour porridge

In this study, the bacterial flora structure, sensory quality and culturable lactic acid bacteria resources of sour porridge in Bayannur area were analyzed. Furthermore, these bacterial sequencing data were combined with the high-throughput sequencing data of Ordos sour porridge to analyze the microbiota differences. MiSeq results showed that Lactiplantibacillus, Acetobacter, Lacticaseibacillus, Lactobacillus, and Limosilactobacillus were the main bacterial genera in sour porridge. Further comparative analysis showed that the average relative abundance of 7 dominant bacteria genera was significantly higher in sour porridge from Bayannur area (P < 0.05). Electronic nose and electronic tongue demonstrated that the primary volatile flavor substances in sour porridge were terpene compounds, and the change in taste characteristics was mainly reflected in the sour taste index. Correlation showed that most of LAB genera and Acetobacter were positively correlated with the formation of aromatic compounds. Pure culture showed that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum were the dominant LAB in sour porridge. Overall, certain differences in the bacterial flora of sour porridge from different regions were identified, with Lactiplantibacillus, Acetobacter, Lacticaseibacillus, Lactobacillus, and Limosilactobacillus being characteristic of sour porridge in Bayannur area. Moreover, some LAB genera and Acetobacter were found to have a positive effect on flavor formation in sour porridge.

The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Flexiplex: a versatile demultiplexer and search tool for omics data.

The process of analyzing high throughput sequencing data often requires the identification and extraction of specific target sequences. This could include tasks, such as identifying cellular barcodes and UMIs in single-cell data, and specific genetic variants for genotyping. However, existing tools, which perform these functions are often task-specific, such as only demultiplexing barcodes for a dedicated type of experiment, or are not tolerant to noise in the sequencing data. To overcome these limitations, we developed Flexiplex, a versatile and fast sequence searching and demultiplexing tool for omics data, which is based on the Levenshtein distance and thus allows imperfect matches. We demonstrate Flexiplex's application on three use cases, identifying cell-line-specific sequences in Illumina short-read single-cell data, and discovering and demultiplexing cellular barcodes from noisy long-read single-cell RNA-seq data. We show that Flexiplex achieves an excellent balance of accuracy and computational efficiency compared to leading task-specific tools. Flexiplex is available at https://davidsongroup.github.io/flexiplex/.