PacBio Single Molecule Real Time Research Articles

Integrated PacBio SMRT and Illumina sequencing uncovers transcriptional and physiological responses to drought stress in whole-plant Nitraria tangutorum

IntroductionNitraria tangutorum Bobr., a prominent xerophytic shrub, exhibits remarkable adaptability to harsh environment and plays a significant part in preventing desertification in northwest China owing to its exceptional drought and salinity tolerance.MethodsTo investigate the drought-resistant mechanism underlying N. tangutorum, we treated 8-week-old seedlings with polyethylene glycol (PEG)-6000 (20%, m/m) to induce drought stress. 27 samples from different tissues (leaves, roots and stems) of N. tangutorum at 0, 6 and 24 h after drought stress treatment were sequenced using PacBio single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to obtain a comprehensive transcriptome.ResultsThe PacBio SMRT sequencing generated 44,829 non-redundant transcripts and provided valuable reference gene information. In leaves, roots and stems, we identified 1162, 2024 and 232 differentially expressed genes (DEGs), respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that plant hormone signaling and mitogen-activated protein kinase (MAPK) cascade played a pivotal role in transmitting stress signals throughout the whole N. tangutorum plant following drought stress. The interconversion of starch and sucrose, as well as the biosynthesis of amino acid and lignin, may represent adaptive strategies employed by N. tangutorum to effectively cope with drought. Transcription factor analysis showed that AP2/ERF-ERF, WRKY, bHLH, NAC and MYB families were mainly involved in the regulation of drought response genes. Furthermore, eight physiological indexes, including content of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), total amino acid and soluble sugar, and activities of three antioxidant enzymes were all investigate after PEG treatment, elucidating the drought tolerance mechanism from physiological perspective. The weighted gene co-expression network analysis (WGCNA) identified several hub genes serve as key regulator in response to drought through hormone participation, ROS cleavage, glycolysis, TF regulation in N. tangutorum.DiscussionThese findings enlarge genomic resources and facilitate research in the discovery of novel genes research in N. tangutorum, thereby establishing a foundation for investigating the drought resistance mechanism of xerophyte.

Full-Length Transcriptome Construction and Systematic Characterization of Virulence Factor-Associated Isoforms in Vairimorpha (Nosema) Ceranae.

Vairimorpha (Nosema) ceranae is a single-cellular fungus that obligately infects the midgut epithelial cells of adult honeybees, causing bee microsporidiosis and jeopardizing bee health and production. This work aims to construct the full-length transcriptome of V. ceranae and conduct a relevant investigation using PacBio single-molecule real-time (SMRT) sequencing technology. Following PacBio SMRT sequencing, 41,950 circular consensus (CCS) were generated, and 25,068 full-length non-chimeric (FLNC) reads were then detected. After polishing, 4387 high-quality, full-length transcripts were gained. There are 778, 2083, 1202, 1559, 1457, 1232, 1702, and 3896 full-length transcripts that could be annotated to COG, GO, KEGG, KOG, Pfam, Swiss-Prot, eggNOG, and Nr databases, respectively. Additionally, 11 alternative splicing (AS) events occurred in 6 genes were identified, including 1 alternative 5' splice-site and 10 intron retention. The structures of 225 annotated genes in the V. ceranae reference genome were optimized, of which 29 genes were extended at both 5' UTR and 3' UTR, while 90 and 106 genes were, respectively, extended at the 5' UTR as well as 3' UTR. Furthermore, a total of 29 high-confidence lncRNAs were obtained, including 12 sense-lncRNAs, 10 lincRNAs, and 7 antisense-lncRNAs. Taken together, the high-quality, full-length transcriptome of V. ceranae was constructed and annotated, the structures of annotated genes in the V. ceranae reference genome were improved, and abundant new genes, transcripts, and lncRNAs were discovered. Findings from this current work offer a valuable resource and a crucial foundation for molecular and omics research on V. ceranae.

Full-length transcriptome characterization and comparative analysis of Gleditsia sinensis

As an economically important tree, Gleditsia sinensis Lam. is widely planted. A lack of background genetic information on G. sinensis hinders molecular breeding. Based on PacBio single-molecule real-time (SMRT) sequencing and analysis of G. sinensis, a total of 95,183 non-redundant transcript sequences were obtained, of which 93,668 contained complete open reading frames (ORFs), 2,858 were long non-coding RNAs (LncRNAs) and 18,855 alternative splicing (AS) events were identified. Genes orthologous to different Gleditsia species pairs were identified, stress-related genes had been positively selected during the evolution. AGA, AGG, and CCA were identified as the universal optimal codon in the genus of Gleditsia. EIF5A was selected as a suitable fluorescent quantitative reference gene. 315 Cytochrome P450 monooxygenases (CYP450s) and 147 uridine diphosphate (UDP)-glycosyltransferases (UGTs) were recognized through the PacBio SMRT transcriptome. Randomized selection of GsIAA14 for cloning verified the reliability of the PacBio SMRT transcriptome assembly sequence. In conclusion, the research data lay the foundation for further analysis of the evolutionary mechanism and molecular breeding of Gleditsia.

Comparative transcriptome analysis of dioecious floral development in Trachycarpus fortunei using Illumina and PacBio SMRT sequencing

BackgroundTrachycarpus fortunei is a plant with significant economic and ornamental value. Both male and female flowers of T. fortunei originate as bisexual flowers, and selective abortion occurs during floral development. However, the regulatory mechanisms underlying this process remain unclear in T. fortunei. In this study, transcriptome sequencing with Illumina and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) platforms were used to investigate gene expression differences between male and female T. fortunei plants.ResultsA total of 833,137 full-length non-chimeric (FLNC) reads were obtained, and 726,846 high-quality full-length transcripts were identified. A total of 159 genes were differentially expressed between male and female flowers at all development stages. Some of the differentially expressed genes (DEGs) showed male bias, including serine/threonine-protein kinase (STPK), THUMP1 homolog and other genes. Through single-nucleotide polymorphisms(SNPs) identification, 28 genes were considered as potential sex-associated SNPs. Time-Ordered Gene Co-expression Network (TO-GCN) analysis revealed that MADS2 and MADS26 may play important roles in the development of female and male flowers T. fortune plants, respectively.ConclusionsThese findings provide a genetic basis for flower development and differentiation in T. fortunei, and improve our understanding of the mechanisms underlying sexual differentiation in T. fortunei.

Long-read assembled metagenomic approaches improve our understanding on metabolic potentials of microbial community in mangrove sediments

BackgroundMangrove wetlands are coastal ecosystems with important ecological features and provide habitats for diverse microorganisms with key roles in nutrient and biogeochemical cycling. However, the overall metabolic potentials and ecological roles of microbial community in mangrove sediment are remained unanswered. In current study, the microbial and metabolic profiles of prokaryotic and fungal communities in mangrove sediments were investigated using metagenomic analysis based on PacBio single-molecule real time (SMRT) and Illumina sequencing techniques.ResultsComparing to Illumina short reads, the incorporation of PacBio long reads significantly contributed to more contiguous assemblies, yielded more than doubled high-quality metagenome-assembled genomes (MAGs), and improved the novelty of the MAGs. Further metabolic reconstruction for recovered MAGs showed that prokaryotes potentially played an essential role in carbon cycling in mangrove sediment, displaying versatile metabolic potential for degrading organic carbons, fermentation, autotrophy, and carbon fixation. Mangrove fungi also functioned as a player in carbon cycling, potentially involved in the degradation of various carbohydrate and peptide substrates. Notably, a new candidate bacterial phylum named as Candidatus Cosmopoliota with a ubiquitous distribution is proposed. Genomic analysis revealed that this new phylum is capable of utilizing various types of organic substrates, anaerobic fermentation, and carbon fixation with the Wood-Ljungdahl (WL) pathway and the reverse tricarboxylic acid (rTCA) cycle.ConclusionsThe study not only highlights the advantages of HiSeq-PacBio Hybrid assembly for a more complete profiling of environmental microbiomes but also expands our understanding of the microbial diversity and potential roles of distinct microbial groups in biogeochemical cycling in mangrove sediment.-S53N_3zEPEL_LmTMdKdCcVideo

Cellulase–lactic acid bacteria synergy action regulates silage fermentation of woody plant

BackgroundFeed shortage is an important factor limiting livestock production in the world. To effectively utilize natural woody plant resources, we used wilting and microbial additives to prepare an anaerobic fermentation feed of mulberry, and used PacBio single-molecule real-time (SMRT) sequencing technology to analyse the “enzyme–bacteria synergy” and fermentation mechanism.ResultsThe fresh branches and leaves of mulberry have high levels of moisture and nutrients, and also contain a diverse range of epiphytic microorganisms. After ensiling, the microbial diversity decreased markedly, and the dominant bacteria rapidly shifted from Gram-negative Proteobacteria to Gram-positive Firmicutes. Lactic acid bacteria (LAB) emerged as the dominant microbial population, resulting in increased in the proportion of the carbohydrate metabolism and decreased in the proportion of the amino acid and “global and overview map” (GOM) metabolism categories. The combination of cellulase and LAB exhibited a synergistic effect, through which cellulases such as glycanase, pectinase, and carboxymethyl cellulase decomposed cellulose and hemicellulose into sugars. LAB converted these sugars into lactic acid through the glycolytic pathway, thereby improving the microbial community structure, metabolism and fermentation quality of mulberry silage. The GOM, carbohydrate metabolism, and amino acid metabolism were the main microbial metabolic categories during ensiling. The presence of LAB had an important effect on the microbial community and metabolic pathways during silage fermentation. A “co-occurrence microbial network” formed with LAB, effectively inhibiting the growth of harmful microorganisms, and dominating the anaerobic fermentation process.ConclusionsIn summary, PacBio SMRT was used to accurately analyse the microbial network information and regulatory mechanism of anaerobic fermentation, which provided a scientific basis for the study of woody silage fermentation theory. This study reveals for the first time the main principle of the enzyme–bacteria synergy in a woody silage fermentation system, which provides technical support for the development and utilization of woody feed resources, and achieves sustainable livestock production.

Chromosome-level assembly of Dictyophora rubrovolvata genome using third-generation DNA sequencing and Hi-C analysis.

Dictyophora rubrovolvata, a rare edible mushroom with both nutritional and medicinal values, was regarded as the "queen of the mushroom" for its attractive appearance. Dictyophora rubrovolvata has been widely cultivated in China in recent years, and many researchers were focusing on its nutrition, culture condition, and artificial cultivation. Due to a lack of genomic information, research on bioactive substances, cross breeding, lignocellulose degradation, and molecular biology is limited. In this study, we report a chromosome-level reference genome of D. rubrovolvata using the PacBio single-molecule real-time-sequencing technique and high-throughput chromosome conformation capture (Hi-C) technologies. A total of 1.83 Gb circular consensus sequencing reads representing ∼983.34 coverage of the D. rubrovolvata genome were generated. The final genome was assembled into 136 contigs with a total length of 32.89 Mb. The scaffold and contig N50 length were 2.71 and 2.48 Mb, respectively. After chromosome-level scaffolding, 11 chromosomes with a total length of 28.24 Mb were constructed. Genome annotation further revealed that 9.86% of the genome was composed of repetitive sequences, and a total of 508 noncoding RNA (rRNA: 329, tRNA: 150, ncRNA: 29) were annotated. In addition, 9,725 protein-coding genes were predicted, among which 8,830 (90.79%) genes were predicted using homology or RNA-seq. Benchmarking Universal Single-Copy Orthologs results further revealed that there were 80.34% complete single-copy fungal orthologs. In this study, a total of 360 genes were annotated as belonging to the carbohydrate-active enzymes family. Further analysis also predicted 425 cytochromes P450 genes, which can be classified into 41 families. This highly accurate, chromosome-level reference genome of D. rubrovolvata will provide essential genomic information for understanding the molecular mechanism in its fruiting body formation during morphological development and facilitate the exploitation of medicinal compounds produced by this mushroom.

Long-read sequencing improves diagnostic rate in neuromuscular disorders.

Massive parallel sequencing methods, such as exome, genome, and targeted DNA sequencing, have aided molecular diagnosis of genetic diseases in the last 20 years. However, short-read sequencing methods still have several limitations, such inaccurate genome assembly, the inability to detect large structural variants, and variants located in hard-to-sequence regions like highly repetitive areas. The recently emerged PacBio single-molecule real-time (SMRT) and Oxford nanopore technology (ONT) long-read sequencing (LRS) methods have been shown to overcome most of these technical issues, leading to an increase in diagnostic rate. LRS methods are contributing to the detection of repeat expansions in novel disease-causing genes (e.g., ABCD3, NOTCH2NLC and RILPL1 causing an Oculopharyngodistal myopathy or PLIN4 causing a Myopathy with rimmed ubiquitin-positive autophagic vacuolation), of structural variants (e.g., in DMD), and of single nucleotide variants in repetitive regions (TTN and NEB). Moreover, these methods have simplified the characterization of the D4Z4 repeats in DUX4, facilitating the diagnosis of Facioscapulohumeral muscular dystrophy (FSHD). We review recent studies that have used either ONT or PacBio SMRT sequencing methods and discuss different types of variants that have been detected using these approaches in individuals with neuromuscular disorders.

Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing protocols.

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) genome. Single‐molecule real‐time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole‐genome sequencing (WGS) of SARS‐CoV‐2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3–99.5]) than with the amplicon protocol (99.3% [IQR: 69.9–99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for C t values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on C t values, higher S gene coverage with the target capture protocol was observed only for samples with C t > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS‐CoV‐2.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals P450 genes involved in thiamethoxam detoxification in Bradysia odoriphaga

The sciarid fly Bradysia odoriphaga is a serious pest of Chinese chive (Liliaceae). Neonicotinoid insecticides including thiamethoxam have been used for B. odoriphaga control. However, thiamethoxam resistance in B. odoriphaga has developed in recent years. To identify potential genes involved in detoxification metabolism of thiamethoxam in B. odoriphaga, a PacBio single-molecule real-time (SMRT) transcriptome sequencing and Illumina RNA-seq analysis on thiamethoxam treated B. odoriphaga were performed to explore differentially expressed genes in B. odoriphaga. After SMRT sequencing, analysis of Illumina RNA-Seq data showed a total of 172 differentially expressed genes (DEGs) after thiamethoxam treatment, among which eight upregulated DEGs were P450 genes that may be related to thiamethoxam metabolism. The qRT-PCR results of the eight up-regulated P450 unigenes after thiamethoxam treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of the eight upregulated P450 transcripts followed by insecticide bioassay was conducted, and three P450 unigenes were verified to be related to thiamethoxam detoxification in B. odoriphaga. This study provides new information about the P450 genes involved in thiamethoxam detoxification in B. odoriphaga.

Using PacBio SMRT Sequencing Technology and Metabolomics to Explore the Microbiota-Metabolome Interaction Related to Silage Fermentation of Woody Plant

Silage fermentation is a dynamic process involving the succession of microbial communities and changes in metabolites. Fresh branched and leaves of paper mulberry were used to prepared silage. Crop by-products including corn bran, rice bran, and wheat bran were used as exogenous additives. Pacific Biosciences single-molecule real-time (SMRT) sequencing technology and metabolomics are used to explore the interaction mechanism of microbial structure and metabolites during woody silage fermentation and to verify the principle that exogenous additives can modulate silage fermentation. Under the dual stress of anaerobic and acidic environment of silage fermentation, the microbial community changed from Gram-negative to Gram-positive bacteria, and a large amount of lactic acid and volatile fatty acid were produced, which lowered the pH value and caused the rapid death of aerobic bacteria with thin cell walls. The exogenous additives of corn bran and wheat bran accelerated the dynamic succession of lactic acid bacteria as the dominant microbial community in silage fermentation, increased the metabolic pathways of lactic acid, unsaturated fatty acids, citric acid, L-malic acid and other flavoring agents, and inhibited the growth of Clostridium and Enterobacter, thereby improving the flavor and quality of the silage. However, because rice bran contained butyric acid spore bacteria, it can multiply in an anaerobic environment, led to butyric acid fermentation, and promoted protein degradation and ammonia nitrogen production, thereby reduced the fermentation quality of woody silage. The results showed that during the silage fermentation process, the microbial community and the metabolome can interact, and exogenous additives can affect the fermentation quality of silage. SMRT sequencing technology and untargeted metabolomics revealed the microbiota-metabolome interaction during silage fermentation. Changes in the structure of the microbial community can affect the metabolic pathways, and the final metabolites can inhibit the growth of microorganisms that are not conducive to silage fermentation. Exogenous carbohydrate additives can change the fermentation substrate and affect microbial community structure, thus modulate the silage fermentation.

PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus

The CYP2D6 enzyme is estimated to metabolize 25% of commonly used pharmaceuticals and is of intense pharmacogenetic interest due to the polymorphic nature of the CYP2D6 gene. Accurate allele typing of CYP2D6 has proved challenging due to frequent copy number variants (CNVs) and paralogous pseudogenes. SNP-arrays, qPCR and short-read sequencing have been employed to interrogate CYP2D6, however these technologies are unable to capture longer range information. Long-read sequencing using the PacBio Single Molecule Real Time (SMRT) sequencing platform has yielded promising results for CYP2D6 allele typing. However, previous studies have been limited in scale and have employed nascent data processing pipelines. We present a robust data processing pipeline “PLASTER” for accurate allele typing of SMRT sequenced amplicons. We demonstrate the pipeline by typing CYP2D6 alleles in a large cohort of 377 Solomon Islanders. This pharmacogenetic method will improve drug safety and efficacy through screening prior to drug administration.

Microbiome Diversity Analysis of the Bacterial Community in Idah River, Kogi State, Nigeria

The analysis of bacterial diversity in aquatic systems particularly in rivers, lakes, and streams can provide useful data on the effect of anthropogenic activities on such water bodies to humans and fishes. Idah River, the focal point of this study, is an offshoot of the two major Nigerian rivers characterized by observed human activities and pollution sources. Water samples were collected from four designated sites and assessed for their bacterial assemblages and structure, using PacBio Single-Molecule Real-Time (SMRT) sequencing technology. The full length of the 16S rRNA gene was sequenced, and Amplicon Sequence Variants were generated using the DADA2 workflow optimised for PacBio long-read amplicons in Rstudio. A total of 8751 high-quality reads obtained were taxonomically classified as 24 phyla, 42 classes, 84 orders, 125 families, 156 genera, and 106 species. Taxonomical composition revealed Proteobacteria as the most abundant phyla across all sample sites. At the genera level, Azospira (57.03%) was the most dominant ASV in Docking Point A, while Acinetobacter (66.67%) was the most abundant ASV in Docking Point B. In Idah Axis Confluence, hgcl clade (65.66%) was the most prevalent ASV, whereas Holophaga (42.86%) was the most common ASV in Idah Axis Midstream. Genera analysis also revealed that 12.9% of the total ASVs were discovered across all sample sites. Among these were pathogenic bacteria, reducers, and degraders of domestic and animal wastes. Observed results provide evidence that sampled sites of Idah River are contaminated, most likely through constant human activities and thus, could have an impact on resident fishes as well. This study, therefore, agrees with a previous report from the river, which used standard microbial procedures. However, next-generation sequencing techniques employed revealed more bacterial community than the former, including unresolved taxonomic sequences that may be novel.

Characterization of the early gene expression profile in Populus ussuriensis under cold stress using PacBio SMRT sequencing integrated with RNA-seq reads.

Populus ussuriensis is an important and fast-growing afforestation plant species in north-eastern China. The whole-genome sequencing of P. ussuriensis has not been completed. Also, the transcriptional network of P. ussuriensis response to cold stress remains unknown. To unravel the early response of P. ussuriensis to chilling (3 °C) stress and freezing (-3 °C) stresses at the transcriptional level, we performed single-molecule real-time (SMRT) and Illumina RNA sequencing for P. ussuriensis. The SMRT long-read isoform sequencing led to the identification of 29,243,277 subreads and 575,481 circular consensus sequencing reads. Approximately 50,910 high-quality isoforms were generated, and 2272 simple sequence repeats and 8086 long non-coding RNAs were identified. The Ca2+ content and abscisic acid (ABA) content in P. ussuriensis were significantly increased under cold stresses, while the value in the freezing stress treatment group was significantly higher than the chilling stress treatment group. A total of 49 genes that are involved in the signal transduction pathways related to perception and transmission of cold stress signals, such as the Ca2+ signaling pathway, ABA signaling pathway and MAPK signaling cascade, were found to be differentially expressed. In addition, 158 transcription factors from 21 different families, such as MYB, WRKY and AP2/ERF, were differentially expressed during chilling and freezing treatments. Moreover, the measurement of physiological indicators and bioinformatics observations demonstrated the altered expression pattern of genes involved in reactive oxygen species balance and the sugar metabolism pathway during chilling and freezing stresses. This is the first report of the early responses of P. ussuriensis to cold stress, which lays the foundation for future studies on the regulatory mechanisms in cold-stress response. In addition the full-length reference transcriptome of P. ussuriensis deciphered could be used in future studies on P. ussuriensis.

SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

Grazing and Supplementation of Dietary Yeast Probiotics Shape the Gut Microbiota and Improve the Immunity of Black Fattening Goats (Capra hircus).

This study aimed to investigate the effects of different feeding modes on the growth performance, gut microbiota, and immunity of Black Fattening Goat (Capra hircus). A total of 30 goats were grouped in three groups by their feeding modes (pasture grazing group, PG; barn feeding group, BF; barn feeding + probiotics, BF + P; n = 10) and the study was performed for 114 days. After a 2-week adaptation period, the first growth performance test was conducted, and the blood and fecal samplings (day 0) were collected on January 17, 2020, while the second and third test and samplings were conducted on days 53 and 100 of feeding. The species-composition of fecal microbiota was analyzed by 16S ribosomal RNA gene-sequencing using PacBio single molecule real time (SMRT) sequencing technology. Both the BF and BF + P groups had the highest (P < 0.05) body’s weight and length, and chest circumference at days 53 and 100, especially at day 100, the body’s weight of both the BF groups were more than 18 kg. The levels of immunoglobulin A (IgA) and immunoglobulin G (IgG) were found to be significantly higher (P < 0.05) in the PG and BF + P groups at day 100. The PG group exhibited the highest number of operational taxonomic unit (OTUs) and alpha diversity. Firmicutes, Bacteroidetes, and Verrucomicrobia were the predominant phyla in all the fecal samples. The relative abundance of Akkermansia muciniphila and Ruminococcus flavefaciens were found to be significantly higher (P < 0.05) in PG group and BF + P group at day 100, respectively, which might partially explain the significantly higher (P < 0.05) levels of IgA and IgG in these two groups. These findings suggested that BF supplemented with 5 g probiotics (Saccharomyces cerevisiae and mannan oligosaccharides) per day has the potential to enhance the growth and immunity of Black Fattening Goats.

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum.

This study investigated the effect of inoculating Lactobacillus (L.) plantarum PS-8 in fermentation of alfalfa silages. We monitored the fermentation characteristics and bacterial population dynamics during the ensiling process. PacBio single molecule real time sequencing was combined with propidium monoazide (PMA) treatment to monitor the viable microbiota dynamics. We found that inoculating L. plantarum PS-8 may improve the silage quality by accelerating acidification, reducing the amounts of clostridia, coliform bacteria, molds and yeasts, elevating the protein and organic acid contents (except butyrate), and enhancing lactic acid bacteria (LAB) while suppressing harmful microorganisms. Some significant differential abundant taxa were found between the PMA-treated and non-treated microbiota. For example, the relative abundances of L. brevis, L. plantarum, and Pediococcus pentosaceus were significantly higher in the PMA-treated group than the non-PMA-treated group, suggesting obvious differences between the viable and non-viable microbiota. It would thus be necessary to distinguish between the viable and non-viable microbial communities to further understand their physiological contribution in silage fermentation. By tracking the dynamics of viable microbiota in relation with changes in the physico-chemical parameters, our study provided novel insights into the beneficial effects of inoculating L. plantarum PS-8 in silage fermentation and the physiological function of the viable bacterial communities.

The use of PacBio SMRT technology to explore the microbial network and fermentation characteristics of woody silage prepared with exogenous carbohydrate additives.

To effectively use woody plant resources to prepare silage for ruminants, Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing was applied to study the microbial network and fermentation characteristics of paper mulberry (PM) silage prepared with corn meal (CM) and rice bran (RB) as exogenous additives. PM is rich in nutrients and contains more than 26% crude protein in dry matter. After ensiling, the microbial diversity and abundance in PM, CM and RB decreased due to the anaerobic environment and acidic conditions. The CM-treated PM silage accelerated the conversion of the dominant microbial community from harmful bacteria to lactic acid bacteria and promoted lactic acid fermentation. When RB was used to treat PM silage, Enterobacter and Clostridium species became the main bacterial community during ensiling, leading to butyric acid fermentation and protein decomposition. Compared with RB, CM increased the amount of fermentation substrates, changed the microbial community structure and affected metabolic pathways (global metabolism, carbohydrate metabolism and amino acid metabolism), which improved the flavour and quality of the PM silage. The CM addition of improved the fermentation quality of PM silage, with PM + CM being the ideal combination. The SMRT sequencing technology could accurately obtain specific details of the microbial networks and fermentation characteristics. Our results indicate that PM can be used as a potential high-protein silage in animal production. In tropics, the effective use of abundant natural biomass resources such as woody plants to prepare silage for feed preservation can solve the problem of restricting livestock production due to the shortage of feed in the dry season. SMRT sequencing technology was used to accurately analyze the microbial network and fermentation characteristics of woody silage prepared with CM as an exogenous additive to improve the fermentation quality of silage.

Exploring microbial community structure and metabolic gene clusters during silage fermentation of paper mulberry, a high-protein woody plant

To develop a new high-protein woody forage resource for livestock to alleviate feed shortages in the tropics, we applied PacBio single-molecule, real-time (SMRT) sequencing to explore the community structure, species diversity and metabolic gene clusters of natural microorganisms associated with paper mulberry (PM) silage fermentation. High levels of microbial diversity and abundance were observed in PM raw material, and these levels decreased with the progression of silage fermentation. During woody ensiling, the dominant bacteria shifted from pathogenic Gram-negative Proteobacteria to beneficial Gram-positive Firmicutes. Lactic acid bacteria became the most dominant bacteria that affected fermentation quality in terminal silages. Global and overview maps, carbohydrate metabolism and amino-acid metabolism were the important microbial metabolic pathways that impacted the final fermentation product of silage. PacBio SMRT sequencing revealed specific microbial-related information concerning silage. PM is rich in nutrients and macro mineral contents, which are preserved well during ensiling, indicating that PM silage can serve as a new woody resource suitable for ruminants.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals candidate genes and pathway involved in selenium metabolism in hyperaccumulator Cardamine violifolia

BackgroundCardamine violifolia, native to China, is one of the selenium (Se) hyperaccumulators. The mechanism of Se metabolism and tolerance remains unclear, and only limited genetic information is currently available. Therefore, we combined a PacBio single-molecule real-time (SMRT) transcriptome library and the Illumina RNA-seq data of sodium selenate (Na2SeO4)-treated C. violifolia to further reveal the molecular mechanism of Se metabolism.ResultsThe concentrations of the total, inorganic, and organic Se in C. violifolia seedlings significantly increased as the Na2SeO4 treatment concentration increased. From SMRT full-length transcriptome of C. violifolia, we obtained 26,745 annotated nonredundant transcripts, 14,269 simple sequence repeats, 283 alternative splices, and 3407 transcription factors. Fifty-one genes from 134 transcripts were identified to be involved in Se metabolism, including transporter, assimilatory enzyme, and several specific genes. Analysis of Illumina RNA-Seq data showed that a total of 948 differentially expressed genes (DEGs) were filtered from the four groups with Na2SeO4 treatment, among which 11 DEGs were related to Se metabolism. The enrichment analysis of KEGG pathways of all the DEGs showed that they were significantly enriched in five pathways, such as hormone signal transduction and plant-pathogen interaction pathways. Four genes related to Se metabolism, adenosine triphosphate sulfurase 1, adenosine 5′-phosphosulfate reductase 3, cysteine (Cys) desulfurase 1, and serine acetyltransferase 2, were regulated by lncRNAs. Twenty potential hub genes (e.g., sulfate transporter 1;1, Cys synthase, methionine gamma-lyase, and Se-binding protein 1) were screened and identified to play important roles in Se accumulation and tolerance in C. violifolia as concluded by weighted gene correlation network analysis. Based on combinative analysis of expression profiling and annotation of genes as well as Se speciation and concentration in C. violifolia under the treatments with different Na2SeO4 concentrations, a putative Se metabolism and assimilation pathway in C. violifolia was proposed.ConclusionOur data provide abundant information on putative gene transcriptions and pathway involved in Se metabolism of C. violifolia. The findings present a genetic resource and provide novel insights into the mechanism of Se hyperaccumulation in C. violifolia.