PacBio Single-molecule Real-time Sequencing Research Articles

Pacific bioscience sequence technology: Review

Pacific Biosciences has developed a platform that may sequence one molecule of DNA in a period via the polymerization of that strand with one enzyme. Single-molecule real-time sequencing by Pacific BioSciences’ technology is one of the most widely utilized third-generation sequencing technologies. PacBio single-molecule real-time Sequencing uses the Zero-mode waveguide’s ingenuity to distinguish the best fluorescence signal from the stable fluorescent backgrounds generated by disorganized free-floating nucleotides. PacBio single-molecule real-time sequencing does not require PCR amplification, and the browse length is a hundred times longer than next-generation sequencing. It will only cover high-GC and high-repeat sections and is more accurate in quantifying low-frequency mutations. PacBio single-molecule real-time sequencing will have a relatively high error rate of 10%-15% (which is practically a standard flaw of existing single-molecule sequencing technology). In contrast to next-generation sequencing, however, the errors are unintentionally random. As a result, multiple sequencing will effectively rectify the bottom deviance. Unlike second-generation sequencing, PacBio sequencing may be a technique for period sequencing and doesn’t need an intermission between browse steps. These options distinguish PacBio sequencing from second-generation sequencing, therefore it’s classified because of the third-generation sequencing. PacBio sequencing produces extremely lengthy reads with a high error rate and low yield. Short reads refine alignments/assemblies/detections to single-nucleotide precision, whereas PacBio long reads provide reliable alignments, scaffolds, and approximate detections of genomic variations. Through extraordinarily long sequencing reads (average >10,000 bp) and high accord precision, the PacBio Sequencing System can provide a terribly high depth of genetic information. To measure and promote the event of modern bioinformatics tools for PacBio sequencing information analysis, a good browse machine is required.

Full-length transcriptome analysis of multiple organs and identification of adaptive genes and pathways in Mikania micrantha

Mikania micrantha is a notorious invasive weed that has caused huge economic loss and negative ecological consequences in invaded areas. This species can adapt well to invasive environments with various stress factors. The identification of gene families and functional pathways related to environmental adaptability is lack in M. micrantha at the multi-organ full-length transcriptome level. In this study, we sequenced the transcriptomes of five M. micrantha organs using PacBio single-molecule real-time sequencing and Illumina RNA sequencing technologies. Based on the transcriptome data, full-length transcripts were captured and gene expression patterns among the five organs were analyzed. KEGG enrichment analysis of genes with higher expression indicated their special roles in environmental stress response and adversity adaptation in the various five organs. The gene families and pathways related to biotic and abiotic factors, including terpene synthases, glutathione S-transferases, antioxidant defense system, and terpenoid biosynthesis pathway, were characterized. The expression levels of most differentially expressed genes in the antioxidant defense system and terpenoid biosynthesis pathway were higher in root, stem, and leaf than in the other two organs, suggesting that root, stem, and leaf have strong ability to respond to adverse stresses and form the important organs of terpenoid synthesis and accumulation. Additionally, a large number of transcription factors and alternative splicing events were predicted. This study provides a comprehensive transcriptome resource for M. micrantha, and our findings facilitate further research on the adaptive evolution and functional genomics of this species.

Allele‐aware chromosome‐scale assembly of the allopolyploid genome of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro)

Dendrocalamus latiflorus Munro is a woody clumping bamboo with rapid shoot growth. Both genetic transformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing techniques are available for D. latiflorus, enabling reverse genetic approaches. Thus, D. latiflorus has the potential to be a model bamboo species. However, the genome sequence of D. latiflorus has remained unreported due to its polyploidy and large genome size. Here, we sequenced the D. latiflorus genome and assembled it into three allele-aware subgenomes (AABBCC), representing the largest genome of a major bamboo species. We assembled 70 allelic chromosomes (2, 737 Mb) for hexaploid D. latiflorus using both single-molecule sequencing from the Pacific Biosciences (PacBio) Sequel platform and chromosome conformation capture sequencing (Hi-C). Repetitive sequences comprised 52.65% of the D. latiflorus genome. We annotated 135 231 protein-coding genes in the genome based on transcriptomes from eight different tissues. Transcriptome sequencing using RNA-Seq and PacBio single-molecule real-time long-read isoform sequencing revealed highly differential alternative splicing (AS) between non-abortive and abortive shoots, suggesting that AS regulates the abortion rate of bamboo shoots. This high-quality hexaploid genome and comprehensive strand-specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D. latiflorus as a model species.

Alleviation of Hepatic Steatosis: Dithizone-Related Gut Microbiome Restoration During Paneth Cell Dysfunction.

Non-alcoholic fatty liver disease (NAFLD), the world’s most common chronic liver disease, is increasingly linked to gut dysbiosis. Paneth cells secrete antimicrobial peptides that regulate the gut microbiome, but their role in the pathogenesis of NAFLD remains unclear. Here, we determine the changes in NAFLD development and gut microbial composition and function via the injection of dithizone that can pharmacologically deplete the granules of Paneth cells. Eight-week-old C57BL/6J male mice (n = 31) were given a high-fat diet (HFD) or standard control diet for 12 weeks. Dithizone (10 mg/kg) was intravenously injected every 3 weeks during the period of diet feeding. Metagenomic DNA was extracted from fecal samples for PacBio Single-Molecule Real-Time sequencing to identify changes in microbial composition and predicted function. We observed dithizone-treated HFD mice, when compared to non-treated HFD mice, to have significant reductions in hepatic triglyceride content (28.98 vs. 53.52 mg/g, p = 0.0419); plasma insulin level (2.18 vs. 6.63 ng/ml, p = 0.0079); and relative mRNA levels of fatty acid synthase (0.52 vs. 1.57, p = 0.0428) and stearoyl-CoA desaturase-1 (0.43 vs. 1.20, p = 0.0121). Bacterial taxonomic profiling found dithizone-treated HFD mice, when compared to non-treated HFD mice, had a lower Firmicutes/Bacteroidetes ratio (2.53 vs. 5.26, p = 0.0541); a higher relative abundance of Bacteroides ASV21 and ASV42 (1.04 vs. 0.22%, p = 0.0277 and 0.96 vs. 0.09%, p = 0.0213); and a reduction in microbes belonging to Firmicutes (all p < 0.05). Bacteroides species correlated positively with predicted microbial functions such as L-methionine (r = 0.54, p = 0.0019) and tetrahydrofolate (r = 0.52, p = 0.0029) biosynthesis. Collectively, dithizone treatment was associated with alleviation in the severity of liver steatosis in HFD mice, possibly through gut microbiome modulation involving the increase in Bacteroides, suggesting microbiome-targeted therapies may have a role in the treatment of NAFLD.

Single Molecule Real-Time Sequencing and Traditional Cultivation Techniques Reveal Complex Community Structures and Regional Variations of Psychrotrophic Bacteria in Raw Milk.

In this study, we investigated the species composition and diversity of psychrotrophic bacteria in raw milk from Heilongjiang, Inner Mongolia, Gansu, Henan, Anhui, Jiangsu, Chongqing, and Hunan provinces in China using traditional cultivation and PacBio Single Molecule Real-Time sequencing methods. The isolated psychrotrophic bacteria were highly diverse, which composed of 21 genera and 59 species. Pseudomonas accounted for 58.9% of the total genera while Stenotrophomonas and Enterococcus were also highly represented (above 5.0%). In particular, P. azotoformans occurred at a level of 16.9% and P. paralactis, P. lactis, E. faecalis, and P. marginalis were present in relatively high proportions (above 4.0%). Regional differences were found significantly among the test regions except samples from Heilongjiang and Inner Mongolia were similar. Additionally, differences were observed between days in Henan, Anhui, and Jiangsu samples. Therefore, control strategies must be implemented on regional and season basis.

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.

Prediction of SARS-CoV-2 Variant Lineages Using the S1-Encoding Region Sequence Obtained by PacBio Single-Molecule Real-Time Sequencing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.

Evaluation of lactic acid bacterial communities in spontaneously-fermented dairy products from Tajikistan, Kyrgyzstan and Uzbekistan using culture-dependent and culture-independent methods

In this study, lactic acid bacteria (LAB) communities in 68 samples of spontaneously-fermented dairy products from Tajikistan, Kyrgyzstan and Uzbekistan were investigated using traditional culture methods combined with PacBio single-molecule real-time (SMRT) sequencing technology. In the 518 LAB strains isolated using the culture-dependent method, the dominant species was Lactobacillus delbrueckii subsp. bulgaricus. A total 75 species of LAB were identified using SMRT sequencing; amongst these Lactobacillus delbrueckii (50.85%) was the predominant species. We compared the LAB communities from different countries (Tajikistan, Kyrgyzstan, Uzbekistan) and different milk sources (fermented cows' milk, fermented mares’ milk, cheese). Our results showed that variation in LAB communities from spontaneously-fermented dairy products from Tajikistan, Kyrgyzstan and Uzbekistan depended on geographical location. These data provide the basis for development and use of LAB resources. Moreover, this study provides new insights that can be applied in starter culture development as well as microbial functionality studies.

Full-length transcriptome sequencing of Heliocidaris crassispina using PacBio single-molecule real-time sequencing

The lack of high-throughput sequencing data makes the research progress of Heliocidaris crassispina slow. Therefore, we used PacBio single-molecule real-time sequencing to generate the first full-length transcriptome. Here, 31,181 isoforms were obtained, with an average length of 2383.20 and a N50 length of 2732 bp. Meanwhile, 764 alternative splicing (AS) events, 5098 long-noncoding RNAs (LncRNAs), 6978 simple sequence repeats (SSRs), and 950 hypothetical transcript factors (TFs) were identified. Moreover, five key innate immune pattern recognition receptors (PRRs), including toll-like receptor (TLR), NACHT domain and leucine-rich repeat (NLR), scavenger receptor cysteine-rich (SRCR), peptidoglycan recognition proteins (PGRP), and gram-negative binding proteins (GNBP), were searched in the transcriptome. In addition, 37 isoforms enriched in KEGG and GO immune systems were also detected. The study provid abundant data support for the current research on H. crassispina.

Long-read assembly of the Chinese indigenous Ningxiang pig genome and identification of genetic variations in fat metabolism among different breeds.

Advances in long-read sequencing technology and genome assembly provide an opportunity to improve the pig genome and reveal the full range of structural variations (SVs) between local Chinese and European pigs. To date, little is known about the genomes of some unique Chinese indigenous breeds, such as the Ningxiang pig. Here, we report the sequencing and assembly of a highly contiguous Ningxiang pig genome (NX) via an integration of PacBio single-molecule real-time sequencing, Illumina next-generation sequencing, BioNano optical mapping and Hi-C (chromosome conformation capture) approaches. The assembled genome comprises 2.44Gb with a contig N50 of 26.1Mb and 418 contigs in total. These contigs are organized into 121 scaffolds with a scaffold N50 of 139.0Mb. More than 99.1% of the assembled sequence could be localized to 19 pseudochromosomes and is annotated with 20,914 protein-coding genes and 34.04% repetitive sequences. Comparisons between the NX and European Duroc assemblies revealed many SVs in genes involved in the immune system, nervous system, lipid metabolism and environmental adaptation. The genetic variants include 47 Chinese domestic pig-specific SVs and the associated 74 genes may contribute to the differences in domestic traits compared to European pigs. Moreover, single nucleotide polymorphisms (SNPs) identified from whole genome resequencing data of 73 Chinese pigs, representing 17 geographically isolated breeds, showed their specific genetic variations, population structure and evolutionary patterns. Finally, we explore transcriptional regulation in the first intron of the MYL4 gene, as the genomic SV (281-bp deletion) in Ningxiang pig promotes its subcutaneous fat compared to European pig breeds. This work identifies a set of Asian-specific SVs and SNPs, which will be important resources for modern pig breeding and genetic conservation.

Single-molecule real-time transcript sequencing of developing cotton anthers facilitates genome annotation and fertility restoration candidate gene discovery

Heterosis refers to the superior phenotypes observed in hybrids. Cytoplasmic male sterility (CMS) system plays an important role in cotton heterosis utilization. However, the global gene expression patterns of CMS-D2 and its interaction with the restorer gene Rf1 remain unclear. Here, the full-length transcript sequencing was performed in anthers of the CMS-D2 restorer line using PacBio single-molecule real-time sequencing technology. Combining PacBio SMRT long-read isoforms and Illumina RNA-seq data, 107,066 isoforms from 44,338 loci were obtained, including 10,086 novel isoforms of novel genes and 66,419 new isoforms of known genes. Totally 56,572 alternative splicing (AS) events, 1146 lncRNAs, 61 fusion transcripts and 10,466 genes exhibited alternative polyadenylation (APA), and 60,995 novel isoforms with predicted open reading frames (ORFs) were further identified. Furthermore, the specifically expressed genes in restorer line were selected and confirmed by qRT-PCR. These findings provide a basis for upland cotton genome annotation and transcriptome research, and will help to reveal the molecular mechanism of interaction between Rf1 and CMS-D2 cytoplasm.

The Mycobacterial DNA Methyltransferase HsdM Decreases Intrinsic Isoniazid Susceptibility.

Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is a serious infectious disease worldwide. Multidrug-resistant TB (MDR-TB) remains a global problem, and the understanding of this resistance is incomplete. Studies suggested that DNA methylation promotes bacterial adaptability to antibiotic treatment, but the role of mycobacterial HsdM in drug susceptibility has not been explored. Here, we constructed an inactivated Mycobacterium bovis (BCG) strain, ΔhsdM. ΔhsdM shows growth advantages over wild-type BCG under isoniazid treatment and hypoxia-induced stress. Using high-precision PacBio single-molecule real-time sequencing to compare the ΔhsdM and BCG methylomes, we identified 219 methylated HsdM substrates. Bioinformatics analysis showed that most HsdM-modified genes were enriched in respiration- and energy-related pathways. qPCR showed that HsdM-modified genes directly affected their own transcription, indicating an altered redox regulation. The use of the latent Wayne model revealed that ΔhsdM had growth advantages over wild-type BCG and that HsdM regulated trcR mRNA levels, which may be crucial in regulating transition from latency to reactivation. We found that HsdM regulated corresponding transcription levels via gene methylation; thus, altering the mycobacterial redox status and decreasing the bacterial susceptibility to isoniazid, which is closely correlated with the redox status. Our results provide valuable insight into DNA methylation on drug susceptibility.

SMRT Sequencing of the Full-Length Transcriptome of the Coelomactra antiquata.

Coelomactra antiquata is an important aquatic economic shellfish with high medicinal value. However, because C. antiquata has no reference genome, a lot of molecular biology research cannot be carried out, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. antiquata. In the present study, we conducted the first full-length transcriptome analysis of C. antiquata by using PacBio single-molecule real-time (SMRT) sequencing technology. The results identified a total of 39,209 unigenes with an average length of 2,732 bp, 23,338 CDSs, 251 AS events, 9,881 lncRNAs, 20,106 SSRs, and 2,316 TFs. Subsequently, 59.22% (23,220) of the unigenes were successfully annotated, of which 23,164, 18,711, 15,840, 13,534, and 13,474 unigenes could be annotated using NR, Swiss-prot, KOG, GO, and KEGG databases, respectively. This study lays the foundation for the follow-up research of molecular biology and provides a reference for studying the more medicinal value of C. antiquata.

Full-Length SMRT Transcriptome Sequencing and SSR Analysis of Bactrocera dorsalis (Hendel).

Simple SummaryIn this study, a full-length transcriptome was analyzed with single-molecule real-time (SMRT) sequencing, which was first used to discover simple sequence repeat (SSR) genetic markers from B. dorsalis. Moreover, SSR markers from isoforms were screened for the identification of species diversity. These results could provide molecular biology methods for further population research.Bactrocera dorsalis (Hendel), as one of the most notorious and destructive invasive agricultural pests in the world, causes damage to over 250 different types of fruits and vegetables throughout tropical and subtropical areas. PacBio single-molecule real-time (SMRT) sequencing was used to generate the full-length transcriptome data of B. dorsalis. A total of 40,319,890 subreads (76.6 Gb, clean reads) were generated, including 535,241 circular consensus sequences (CCSs) and 386,916 full-length non-concatemer reads (FLNCs). Transcript cluster analysis of the FLNC reads revealed 22,780 high-quality reads (HQs). In total, 12,274 transcripts were functionally annotated based on four different databases. A total of 1978 SSR loci were distributed throughout 1714 HQ transcripts, of which 1926 were complete SSRs and 52 were complex SSRs. Among the total SSR loci, 2–3 nucleotide repeats were dominant, occupying 83.62%, of which di- and tri- nucleotide repeats were 39.38% and 44.24%, respectively. We detected 105 repeat motifs, of which AT/AT (50.19%), AC/GT (39.15%), CAA/TTG (32.46%), and ACA/TGT (10.86%) were the most common in di- and tri-nucleotide repeats. The repeat SSR motifs were 12–190 bp in length, and 1638 (88.02%) were shorter than 20 bp. According to the randomly selected microsatellite sequence, 80 pairs of primers were designed, and 174 individuals were randomly amplified by PCR using primers. The number of primers that had amplification products with clear bands and showed good polymorphism came to 41, indicating that this was a feasible way to explore SSR markers from the transcriptomic data of B. dorsalis. These results lay a foundation for developing highly polymorphic microsatellites for researching the functional genomics, population genetic structure, and genetic diversity of B. dorsalis.

Discovery of alternatively spliced isoforms and long non-coding RNA in full length brain transcriptomes of anadromous Hilsa shad, Tenualosa ilisha (Hamilton, 1822).

Full length transcriptomes, achieved through long-read sequencing, along with the isoform analysis can reveal complexities in the gene expression profiles, as well as annotate the transcriptomes of non-model organisms. Full length transcripts of brain transcriptome of Tenualosa ilisha, Hilsa shad, were generated through PacBio single molecule real-time sequencing and were characterized. A total of 8.30 Gb clean reads were generated, with PacBio RSII, which resulted in 57,651 high quality consensus transcripts. After removing redundant reads, a total of 19,220 high-quality non-redundant transcripts and 17,341 full length ORF transcripts were classified to 7522 putative ortholog groups. Genes involved in various neural pathways were identified. In addition, isoform clusters and lncRNAs were discovered, along with Hilsa specific transcripts with coding frames and 29,147 SSRs in 944 transcripts (1141 annotated). The present study provided, for the first time, a comprehensive view of the alternative isoforms of genes and transcriptome complexity in Hilsa shad brain and forms a rich resource for functional studies in brain of this anadromous fish.

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.

Full-length transcriptome analysis of Spodoptera frugiperda larval brain reveals detoxification genes.

BackgroundSpodoptera frugiperda (J. E. Smith), commonly known as fall armyworm (FAW), is one of the most destructive agricultural pests in the world and has posed a great threat to crops. The improper use of insecticides has led to rapid development of resistance. However, the genetic data available for uncovering the insecticide resistance mechanisms are scarce.MethodsIn this study, we used PacBio single-molecule real-time (SMRT) sequencing aimed at revealing the full-length transcriptome profiling of the FAW larval brain to obtain detoxification genes.ResultsA total of 18,642 high-quality transcripts were obtained with an average length of 2,371 bp, and 11,230 of which were successfully annotated in six public databases. Among these, 5,692 alternative splicing events were identified.

Microbial community structure, co-occurrence network and fermentation characteristics of woody plant silage.

Feed shortage is a factor restricting animal production in the tropics, therefore how to use natural woody plant resources as animal feed is an important strategy. Under the dual stress of an anaerobic and acidic environment, the microbial response during the fermentation of paper mulberry (PM) silage was found to be sensitive. The Gram-negative bacteria and mould died, and the dominant microbial community rapidly shifted to Gram-positive bacteria, resulting in a large reduction in microbial diversity and abundance. Exogenous bran additives interfered with the stress effects of the woody silage environment. Wheat bran (WB) accelerated the response of microorganisms to the anaerobic stress, and lactic acid bacteria became the dominant microbial community, thereby enhancing the lactic acid fermentation of silage, affecting the metabolic pathways of microorganisms, and improving the flavour and quality of the silage. Addition of rice bran made Enterobacter and Clostridium species quickly respond to the stress of the silage environment and become the predominant bacterial groups. In particular, anaerobic and spore-forming Clostridium species showed a strong tolerance to the silage environment, leading to butyric acid fermentation and protein degradation of the silage, and reducing its fermentation quality. The PacBio single-molecule real-time (SMRT) sequencing technology accurately revealed the microbial co-occurrence network and fermentation mechanism of silage. Our results indicate that PM can be used in combination with WB to prepare high-quality silage for animal production. © 2021 Society of Chemical Industry.

PacBio sequencing revealed variation in the microbiota diversity, species richness and composition between milk collected from healthy and mastitis cows.

Mastitis is the economically most important disease of dairy cows. This study used PacBio single-molecule real-time sequencing technology to sequence the full-length 16S rRNAs from 27 milk samples (18 from mastitis and nine from healthy cows; the cows were at different stages of lactation). We observed that healthy or late stage milk microbiota had significantly higher microbial diversity and richness. The community composition of the microbiota of different groups also varied greatly. The healthy cow milk microbiota was predominantly comprised of Lactococcus lactis, Acinetobacter johnsonii, and Bacteroides dorei, while the milk from mastitis cows was predominantly comprised of Bacillus cereus. The prevalence of L. lactis and B. cereus in the milk samples was confirmed by digital droplets PCR. Differences in the milk microbiota diversity and composition could suggest an important role for some these microbes in protecting the host from mastitis while others associated with mastitis. The results of our research serve as useful references for designing strategies to prevent and treat mastitis.

Third-generation sequencing and metabolome analysis reveal candidate genes and metabolites with altered levels in albino jackfruit seedlings

BackgroundMost plants rely on photosynthesis; therefore, albinism in plants with leaves that are white instead of green causes slow growth, dwarfing, and even death. Although albinism has been characterized in annual model plants, little is known about albino trees. Jackfruit (Artocarpus heterophyllus) is an important tropical fruit tree species. To gain insight into the mechanisms underlying the differential growth and development between albino jackfruit mutants and green seedlings, we analyzed root, stem, and leaf tissues by combining PacBio single-molecule real-time (SMRT) sequencing, high-throughput RNA-sequencing (RNA-seq), and metabolomic analysis.ResultsWe identified 8,202 differentially expressed genes (DEGs), including 225 genes encoding transcription factors (TFs), from 82,572 full-length transcripts. We also identified 298 significantly changed metabolites (SCMs) in albino A. heterophyllus seedlings from a set of 692 metabolites in A. heterophyllus seedlings. Pathway analysis revealed that these DEGs were highly enriched in metabolic pathways such as ‘photosynthesis’, ‘carbon fixation in photosynthetic organisms’, ‘glycolysis/gluconeogenesis’, and ‘TCA cycle’. Analysis of the metabolites revealed 76 SCMs associated with metabolic pathways in the albino mutants, including L-aspartic acid, citric acid, succinic acid, and fumaric acid. We selected 225 differentially expressed TF genes, 333 differentially expressed metabolic pathway genes, and 76 SCMs to construct two correlation networks. Analysis of the TF–DEG network suggested that basic helix-loop-helix (bHLH) and MYB-related TFs regulate the expression of genes involved in carbon fixation and energy metabolism to affect light responses or photomorphogenesis and normal growth. Further analysis of the DEG–SCM correlation network and the photosynthetic carbon fixation pathway suggested that NAD-ME2 (encoding a malic enzyme) and L-aspartic acid jointly inhibit carbon fixation in the albino mutants, resulting in reduced photosynthetic efficiency and inhibited plant growth.ConclusionsOur preliminarily screening identified candidate genes and metabolites specifically affected in albino A. heterophyllus seedlings, laying the foundation for further study of the regulatory mechanism of carbon fixation during photosynthesis and energy metabolism. In addition, our findings elucidate the way genes and metabolites respond in albino trees.