Complete genome sequence of Bacillus stercoris BST19, a soil isolate with potential anti-microbial properties using PacBio sequencing.

We report the complete genome sequence of Bacillus stercoris BST19, an isolate from the allotment soil in Tainan, Taiwan. The genome was obtained using the PacBio Sequel II platform, yielding a circular chromosome of 4,167,147 bp with a 43.9% GC content.

Exploring the cobia (Rachycentron canadum) genome: unveiling putative male heterogametic regions and identification of sex-specific markers.

BACKGROUND: Cobia (Rachycentron canadum) is the only member of the Rachycentridae family and exhibits considerable sexual dimorphism in growth rate. Sex determination in teleosts has been a long-standing basic biological question, and the molecular mechanisms of sex determination/differentiation in cobia are completely unknown. RESULTS: Here, we reported 2 high-quality, chromosome-level annotated male and female cobia genomes with assembly sizes of 586.51 Mb (contig/scaffold N50: 86.0 kb/24.3 Mb) and 583.88 Mb (79.9 kb/22.5 Mb), respectively. Synteny inference among perciform genomes revealed that cobia and the remora Echeneis naucrates were sister groups. Further, whole-genome resequencing of 31 males and 60 females, genome-wide association study, and sequencing depth analysis identified 3 short male-specific regions within a 10.7-kb continuous genomic region on male chromosome 18, which hinted at an undifferentiated sex chromosome system with a putative XX/XY mode of sex determination in cobia. Importantly, the only 2 genes within/between the male-specific regions, epoxide hydrolase 1 (ephx1, renamed cephx1y) and transcription factor 24 (tcf24, renamed ctcf24y), showed testis-specific/biased gene expression, whereas their counterparts cephx1x and ctf24x, located in female chromosome 18, were similarly expressed in both sexes. In addition, male-specific PCR targeting the cephx1y gene revealed that this genomic feature is conserved in cobia populations from Panama, Brazil, Australia, and Japan. CONCLUSION: The first comprehensive genomic survey presented here is a valuable resource for future studies on cobia population structure and dynamics, conservation, and evolutionary history. Furthermore, it establishes evidence of putative male heterogametic regions with 2 genes playing a potential role in the sex determination of the species, and it provides further support for the rapid evolution of sex-determining mechanisms in teleost fish.

Relationship between dynamic changes of microorganisms in Qupi and the quality formation of Fengxiangxing Huairang Daqu.

Introduction: Fengxiangxing Huairang Daqu (FHD) is one of the major types of Daqu in China. However, the relationship between the microbial community structure at different stages, the changes in the sensory characteristics, fermentation characteristics, volatiles, the most critical process point, and the quality formation of FHD is not clear. Methods: Based on microscopic characterization, PacBio SMRT sequencing, and HS-SPME-GC-MS volatile metabolite analysis revealed the relationship between FHD quality formation and the dynamics of Qupi. Results: The results showed that the 12th day of the culture was the most critical process point, highlighting the most significant differences in microbial community structure, sensory characteristics, fermentation characteristics, and flavor substances. Bacillus licheniformis (43.25%), Saccharopolyspora rectivirgula (35.05%), Thermoascus aurantiacus (76.51%), Aspergillus amstelodami (10.81%), and Saccharomycopsis fibuligera (8.88%) were the dominant species in FHD. S. fibuligera, A. amstelodami, and T. aurantiacus were associated with the snow-white color of the FHD epidermis, the yellow color of the interior, and the gray-white color, respectively. The abundance of T. aurantiacus, A. amstelodami, B. licheniformis, and S. rectivirgula was positively associated with the esterifying power and liquefying power of FHD. The abundance of T. aurantiacus and A. amstelodami was positively correlated with the saccharifying power of FHD. The abundance of S. fibuligera was positively related to the fermenting power of FHD. A total of 248 volatiles were detected in Qupi, mainly including alcohols, esters, aldehydes, and ketones. Of them, eleven volatiles had a significant effect on the flavor of Qupi, such as 1-butanol-3-methyl-, hydrazinecarboxamide, ethanol, phenylethyl alcohol, ethyl acetate, 2-octanone, 1-octen-3-ol, formic acid-hexyl ester, (E)-2-octen-1-ol, ethyl hexanoate, and 2(3H)-furanone-dihydro-5-pentyl-. The abundance of B. licheniformis, S. rectivirgula, T. aurantiacus, and S. fibuligera was positively correlated with the alcohols, aromatic compounds, and phenols in FHD. The abundance of S. fibuligera was positively correlated with the acids, esters, and hydrocarbons in FHD. Discussion: These results indicate important theoretical basis and technical support for controllable adjustment of FHD microbial community structure, stable control of FHD quality, and precise, effective, and large-scale guidance of FHD production.

A chromosome-level genome assembly and annotation of the medicinal plant Lepidium apetalum.

OBJECTIVES: As a traditional Chinese medicine, Lepidium apetalum is commonly used for purging the lung, relieving dyspnea, alleviating edema, and has the significant pharmacological effects on cardiovascular disease, hyperlipidemia, etc. In addition, the seeds of L. apetalum are rich in unsaturated fatty acids, sterols, glucosinolates and have a variety of biological activity compounds. To facilitate genomics, phylogenetic and secondary metabolite biosynthesis studies of L. apetalum, we assembled the high-resolution genome of L. apetalum. DATA DESCRIPTION: We completed chromosome-level genome assembly of the L. apetalum genome (2n = 32), using Illumina HiSeq and PacBio Sequel sequencing platform as well as high-throughput chromosome conformation capture (Hi-C) technique. The assembled genome was 296.80 Mb in size, 34.41% in GC content, and 23.89% in repeated sequence content, including 316 contigs with a contig N50 of 16.31 Mb. Hi-C scaffolding resulted in 16 chromosomes occupying 99.79% of the assembled genome sequences. A total of 46 584 genes and 105 pseudogenes were predicted, 98.37% of which can be annotated to Nr, GO, KEGG, TrEMBL, SwissPort, Pfam and KOG databases. The high-quality reference genome generated by this study will provide accurate genetic information for the molecular biology research of L. apetalum.

DNA Isolation Long-Read Genomic Sequencing in Ctenophores.

Long-read sequencing has proven the necessity for high-quality genomic assemblies of reference species, including enigmatic ctenophores. Obtaining high-molecular-weight genomic DNA is pivotal to this process and has proven highly problematic for many species. Here, we discuss different methodologies for gDNA isolation and present a protocol for isolating gDNA for several members of the phylum Ctenophora. Specifically, we describe a Pacific Biosciences library construction method used in conjunction with gDNA isolation methods that have proven successful in obtaining high-quality genomic assemblies in ctenophores.

De novo assembly and annotation of the Patagonian toothfish (Dissostichus eleginoides) genome.

BACKGROUND: Patagonian toothfish (Dissostichus eleginoides) is an economically and ecologically important fish species in the family Nototheniidae. Juveniles occupy progressively deeper waters as they mature and grow, and adults have been caught as deep as 2500 m, living on or in just above the southern shelves and slopes around the sub-Antarctic islands of the Southern Ocean. As apex predators, they are a key part of the food web, feeding on a variety of prey, including krill, squid, and other fish. Despite its importance, genomic sequence data, which could be used for more accurate dating of the divergence between Patagonian and Antarctic toothfish, or establish whether it shares adaptations to temperature with fish living in more polar or equatorial climes, has so far been limited. RESULTS: A high-quality D. eleginoides genome was generated using a combination of Illumina, PacBio and Omni-C sequencing technologies. To aid the genome annotation, the transcriptome derived from a variety of toothfish tissues was also generated using both short and long read sequencing methods. The final genome assembly was 797.8 Mb with a N50 scaffold length of 3.5 Mb. Approximately 31.7% of the genome consisted of repetitive elements. A total of 35,543 putative protein-coding regions were identified, of which 50% have been functionally annotated. Transcriptomics analysis showed that approximately 64% of the predicted genes (22,617 genes) were found to be expressed in the tissues sampled. Comparative genomics analysis revealed that the anti-freeze glycoprotein (AFGP) locus of D. eleginoides does not contain any AFGP proteins compared to the same locus in the Antarctic toothfish (Dissostichus mawsoni). This is in agreement with previously published results looking at hybridization signals and confirms that Patagonian toothfish do not possess AFGP coding sequences in their genome. CONCLUSIONS: We have assembled and annotated the Patagonian toothfish genome, which will provide a valuable genetic resource for ecological and evolutionary studies on this and other closely related species.

Properties and Fungal Communities of Different Soils for Growth of the Medicinal Asian Water Plantain, Alisma orientale, in Fujian, China.

The Asian water plantain, Alisma orientale (Sam.) Juzep, is a traditional Chinese medicinal plant. The dried tubers of the Alisma orientale, commonly referred to as Alismatis rhizome (AR), have long been used in traditional Chinese medicine to treat a variety of diseases. Soil properties and the soil microbial composition are known to affect the quality and bioactivity of plants. Here, we sought to identify variations in soil fungal communities and soil properties to determine which would be optimal for cultivation of A. orietale. Soil properties, heavy metal content, and pesticide residues were determined from soils derived from four different agricultural regions around Shaowu City, Fujian, China, that had previously been cultivated with various crops, namely, Shui Dao Tu (SDT, rice), Guo Shu Tu (GST, pecan), Cha Shu Tu (CST, tea trees), and Sang Shen Tu (SST, mulberry). As fungi can either positively or negatively impact plant growth, the fungal communities in the different soils were characterized using long-read PacBio sequencing. Finally, we examined the quality of A. orientale grown in the different soils. Our results show that fungal community diversity of the GST soil was the highest with saprotrophs the main functional modes in these and SDT soils. Our data show that GST and SDT soils were most suitable for A. orientale growth, with the quality of the AR tubers harvested from GST soil being the highest. These data provide a systematic approach at soil properties of agricultural lands in need of replacement and/or rotating crops. Based on our findings, GST was identified as the optimal soil for planting A. orientale, providing a new resource for local farmers.

Bioturbation effect of artificial inoculation on the flavor metabolites and bacterial communities in the Chinese Mao-tofu fermentation.

A comparison between artificially inoculated Mao-tofu (CC) and naturally fermented Mao-tofu (MM) indicated that artificially adding Mucor plasmaticus to Mao-tofu dramatically enhanced the essential amino acid (EAA) content, as well as umami and sweet amino acids. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis revealed that phenol (3.226 µg/g), 1-octen-3-ol (5.031 µg/g), ethyl heptanoate (1.646 µg/g), and indole (3.422 µg/g) were the key flavor components in Mao-tofu. Unlike MM, CC displayed a substantial increase in esters and a considerable decrease in foul odor substances, including sulfur-containing compounds and indole. Lactococcus raffinolactis, Enterobacter sp. 638, and Streptococcus parauberis KCTC 11537 represented the key bacterial species altering the amino acids and flavor of Mao-tofu according to PacBio single-molecule real-time (SMRT) sequencing and correlation analysis. This study presents the technical feasibility of artificially inoculating Mao-tofu to regulate the core bacterial communities and control the quality of fermented soybean products.

Postmortem Muscle Proteome Characteristics of Silver Carp (Hypophthalmichthys molitrix): Insights from Full-Length Transcriptome and Deep 4D Label-Free Proteomic.

The coverage of the protein database directly determines the results of shotgun proteomics. In this study, PacBio single-molecule real-time sequencing technology was performed on postmortem silver carp muscle transcripts. A total of 42.43 Gb clean data, 35,834 nonredundant transcripts, and 15,413 unigenes were obtained. In total, 99.32% of the unigenes were successfully annotated and assigned specific functions. PacBio long-read isoform sequencing (Iso-Seq) analysis can provide more accurate protein information with a higher proportion of complete coding sequences and longer lengths. Subsequently, 2671 proteins were identified in deep 4D proteomics informed by a full-length transcriptomics technique, which has been shown to improve the identification of low-abundance muscle proteins and potential protein isoforms. The feature of the sarcomeric protein profile and information on more than 30 major proteins in the white dorsal muscle of silver carp were reported here for the first time. Overall, this study provides valuable transcriptome data resources and the comprehensive muscle protein information detected to date for further study into the processing characteristic of early postmortem fish muscle, as well as a spectral library for data-independent acquisition and data processing. This batch of muscle-specific dependent acquisition data is available via PRIDE with identifier PXD043702.

Reference Genome Resource for the Citrus Pathogen Phytophthora citrophthora.

Phytophthora citrophthora is an oomycete pathogen that infects citrus. Its occurrence in citrus-growing regions worldwide is considered a major contributor to crop losses. This study presents a high-quality genome resource for P. citrophthora, which was generated using PacBio HiFi long-read high-throughput sequencing technology. We successfully assembled a 48.5 Mb genome containing 16,409 protein-coding genes from high-quality reads. This marks the first complete genome assembly of P. citrophthora, providing a valuable resource to enhance the understanding of pathogenic behaviour and fungicide sensitivity of this destructive citrus pathogen.

Synthesis of an autochthonous microbial community by analyzing the core microorganisms responsible for the critical flavor of bran vinegar.

Traditional bran vinegar brewing unfolds through natural fermentation, a process driven by spontaneous microbial activity. The unique metabolic activities of various microorganisms lead to distinct flavors and qualities in each batch of vinegar, making it challenging to consistently achieve the desired characteristic flavor compounds. Therefore, identifying the critical microbial species responsible for flavor production and designing starter cultures with improved fermentation efficiency and characteristic flavors are effective methods to address this discrepancy. In this study, 11 core functional microbial species affecting the fermentation flavor of Sichuan shai vinegar (Cupei were placed outside solarization and night-dew for more than one year, and vinegar was the liquid leached from Cupei) (SSV), were revealed by combining PacBio full-length diversity sequencing based on previous metagenomics. The effects of environmental factors and microbial interactions on the growth of 11 microorganisms during fermentation were verified using fermentation experiments. Ultimately, the microbial community was strategically synthesized using a 'top-down' approach, successfully replicating the distinctive flavor profile of Sichuan shai vinegar (SSV). The results showed that the interaction between microorganisms and environmental factors affected microorganism growth. Compared with traditional fermentation, the synthetic microbial community's vinegar-fermented grains (Cupei) can reproduce the key flavor of SSV and is conducive to the production of amino acids. In this study, the key flavor of SSV was reproduced through rational design of the synthetic microbial community. This achievement holds profound significance for the broader application of microbiome assembly strategies in the realm of fermented foods.

Genomic characterization of Bordetella pertussis in South Africa, 2015-2019.

Pertussis remains a public health concern in South Africa, with an increase in reported cases and outbreaks in recent years. Whole genome sequencing was performed on 32 Bordetella pertussis isolates sourced from three different surveillance programmes in South Africa between 2015 and 2019. Genome sequences were characterized using multilocus sequence typing, vaccine antigen genes (ptxP, ptxA, ptxB, prn and fimH) and overall genome structure. All isolates were sequence type 2 and harboured the pertussis toxin promoter allele ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9 %), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Amongst 21 isolates yielding closed genome assemblies, eight distinct genome structures were detected, with 61.9 % (13/21) of the isolates exhibiting three predominant structures. Increases in case numbers are probably not due to evolutionary changes in the genome but possibly due to other factors such as the cyclical nature of B. pertussis disease, waning immunity due to the use of acellular vaccines and/or population immunity gaps.

Complete genome sequence of Arthrobacter sp. KFRI-F3372, a strain isolated from Korean soybean paste Doenjang.

Arthrobacter sp. KFRI-F3372 is a Gram-positive bacterium with a high G + C content of 65.7%, which was isolated from Doenjang, a traditional Korean fermented soybean paste. In this report, we introduce the complete genome sequence of Arthrobacter sp. KFRI-F3372.

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

A new Plasmodium vivax reference genome for South American isolates.

BACKGROUND: Plasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering. RESULTS: This study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions. CONCLUSIONS: Our findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent.

PhaseDancer: a novel targeted assembler of segmental duplications unravels the complexity of the human chromosome 2 fusion going from 48 to 46 chromosomes in hominin evolution.

Resolving complex genomic regions rich in segmental duplications (SDs) is challenging due to the high error rate of long-read sequencing. Here, we describe a targeted approach with a novel genome assembler PhaseDancer that extends SD-rich regions of interest iteratively. We validate its robustness and efficiency using a golden-standard set of human BAC clones and in silico-generated SDs with predefined evolutionary scenarios. PhaseDancer enables extension of the incomplete complex SD-rich subtelomeric regions of Great Ape chromosomes orthologous to the human chromosome 2 (HSA2) fusion site, informing a model of HSA2 formation and unravelling the evolution of human and Great Ape genomes.

Beyond the exome: What's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order, and emerging technologies, such as optical genome mapping and long-read DNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to research consortia focused on elucidating the underlying cause of rare unsolved genetic disorders.

Full-length transcriptome analysis provides new insights into the diversity of immune-related genes in the threatened freshwater shellfish Solenaia oleivora.

Solenaia oleivora, a valuable and rare bivalve endemic to China, is becoming a threatened freshwater sepcies. However, the lack of research on its genome and immune system will hinder advances in its conservation and artificial breeding. In this study, we obtained the full-length transcriptome of S. oleivora using PacBio sequencing. A total of 21,415 transcripts with an average length of 1,726 bp were generated. Among these transcripts, 12,084 had coding sequences (CDS), of which 8,639 were annotated in 6 databases. The structure analysis identified 625 transcript factors (TFs), 8,005 long non-coding RNAs (lncRNAs), and 5,288 simple sequences repeat (SSRs). Meanwhile, massive immune genes were identified from the transcriptome of S. oleivora. In terms of non-self-identification, 97 transcripts of pattern recognition receptors (PRRs) were discovered, including peptidoglycan recognition proteins (PGRPs), gram-negative bacteria binding proteins (GNBPs), toll-like receptors (TLRs), scavenger receptors (SRs), galectins (GALs), C-type lectins (CLTs), and fibrinogen-related protein (FREPs). For pathogen elimination, 7 transcripts related to antimicrobial peptides, lysozymes, and lysosomal enzymes were identified. Moreover, 33 complement-associated transcripts were found. This study enriched the genome resources of S. oleivora and provided new insights for the study of the immune system of S. oleivora.

Analysis on the salt tolerance of Nitraria sibirica Pall. based on Pacbio full-length transcriptome sequencing.

KEY MESSAGE: Nitraria sibirica Pall. regulates its tolerance to salt stress mainly by adjusting ion balance, modifying cell wall structure, and activating signal transduction pathways. N. sibirica, as a typical halophyte, can not only effectively restore saline-alkali land, but also has high economic value. However, studies on its salt tolerance at combining molecular and physiological levels were limited. In this study, the salt tolerance of N. sibirica was analyzed based on Pacbio full-length transcriptome sequencing, and the salt tolerance in the physiological level was verified by key genes. The results showed that 89,017 full-length transcripts were obtained, of which 84,632 sequences were annotated. A total of 86,482 coding sequences (CDS) were predicted and 6561 differentially expressed genes (DEGs) were identified. DEGs were significantly enriched in "sodium ion homeostasis", "response to osmotic stress", "reactive oxygen species metabolic process", "defense response by cell wall thickening", "signal transduction", etc. The expression levels for most of these DEGs increased under salt stress. A total of 69 key genes were screened based on weighted gene co-expression network analysis (WGCNA), of which 33 were first reported on salt tolerance. Moreover, NsRabE1c gene with the highest expression level was selected to verify its salt tolerance. Over-expression of NsRabE1c gene enhanced the germination potential and root length of transgenic Arabidopsis thaliana plants without salt treatment as compared to those of Col-0 and AtRabE1c mutant. The expression levels of NsRabE1c decreased in the growth stagnation phase, while significantly increased in the growth recovery phase under salt stress. We predicted that NsRabE1c gene help N. sibirica resist salt stress through the regulation of plant growth. The results of this study deepen the understanding of salinity resistance in N. sibirica.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes.

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5' untranslated region (5'UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.