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September 22, 2019

Investigating bacterial population structure and dynamics in traditional koumiss from Inner Mongolia using single molecule real-time sequencing.

Koumiss is considered as a complete dairy product high in nutrients and with medicinal properties. The bacterial communities involved in production of koumiss play a crucial role in the fermentation cycle. To reveal bacterial biodiversity in koumiss and the dynamics of succession in bacterial populations during fermentation, 22 samples were collected from 5 sampling sites and the full length of the 16S ribosomal RNA genes sequenced using single molecule real-time sequencing technology. One hundred forty-eight species were identified from 82 bacterial genera and 8 phyla. These results suggested that the structural difference in the bacterial community could be attributed to geographical location. The most significant difference in bacterial composition occurred in samples from group D compared with other groups. The sampling location of group D was distant from the city and maintained the primitive local nomadic life. The dynamics of succession in bacterial communities showed that Lactobacillus helveticus increased in abundance from 0 to 9h and reached its peak at 9h and then decreased. In contrast, Enterococcus faecalis, Enterococcus durans, and Enterococcus casseliflavus increased gradually throughout the fermentation process, and reached a maximum after 24h. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Assessment of the physicochemical properties and bacterial composition of Lactobacillus plantarum and Enterococcus faecium-fermented Astragalus membranaceus using single molecule, real-time sequencing technology.

We investigated if fermentation with probiotic cultures could improve the production of health-promoting biological compounds in Astragalus membranaceus. We tested the probiotics Enterococcus faecium, Lactobacillus plantarum and Enterococcus faecium?+?Lactobacillus plantarum and applied PacBio single molecule, real-time sequencing technology (SMRT) to evaluate the quality of Astragalus fermentation. We found that the production rates of acetic acid, methylacetic acid, aethyl acetic acid and lactic acid using E. faecium?+?L. plantarum were 1866.24?mg/kg on day 15, 203.80?mg/kg on day 30, 996.04?mg/kg on day 15, and 3081.99?mg/kg on day 20, respectively. Other production rates were: polysaccharides, 9.43%, 8.51%, and 7.59% on day 10; saponins, 19.6912?mg/g, 21.6630?mg/g and 20.2084?mg/g on day 15; and flavonoids, 1.9032?mg/g, 2.0835?mg/g, and 1.7086?mg/g on day 20 using E. faecium, L. plantarum and E. faecium?+?L. plantarum, respectively. SMRT was used to analyze microbial composition, and we found that E. faecium and L. plantarum were the most prevalent species after fermentation for 3 days. E. faecium?+?L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Our data demonstrated that the SMRT sequencing platform is applicable to quality assessment of Astragalus fermentation.

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September 22, 2019

Transcriptome analysis of distinct cold tolerance strategies in the rubber tree (Hevea brasiliensis)

Natural rubber is an indispensable commodity used in approximately 40,000 products and is fundamental to the tire industry. Among the species that produce latex, the rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg.], a species native to the Amazon rainforest, is the major producer of latex used worldwide. The Amazon Basin presents optimal conditions for rubber tree growth, but the occurrence of South American leaf blight, which is caused by the fungus Microcyclus ulei (P. Henn) v. Arx, limits rubber tree production. Currently, rubber tree plantations are located in scape regions that exhibit suboptimal conditions such as high winds and cold temperatures. Rubber tree breeding programs aim to identify clones that are adapted to these stress conditions. However, rubber tree breeding is time-consuming, taking more than 20 years to develop a new variety. It is also expensive and requires large field areas. Thus, genetic studies could optimize field evaluations, thereby reducing the time and area required for these experiments. Transcriptome sequencing using next-generation sequencing (RNA-seq) is a powerful tool to identify a full set of transcripts and for evaluating gene expression in model and non-model species. In this study, we constructed a comprehensive transcriptome to evaluate the cold response strategies of the RRIM600 (cold-resistant) and GT1 (cold-tolerant) genotypes. Furthermore, we identified putative microsatellite (SSR) and single-nucleotide polymorphism (SNP) markers. Alternative splicing, which is an important mechanism for plant adaptation under abiotic stress, was further identified, providing an important database for further studies of cold tolerance.

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September 22, 2019

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

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September 22, 2019

Transcriptome profiling of two ornamental and medicinal papaver herbs.

The Papaver spp. (Papaver rhoeas (Corn poppy) and Papaver nudicaule (Iceland poppy)) genera are ornamental and medicinal plants that are used for the isolation of alkaloid drugs. In this study, we generated 700 Mb of transcriptome sequences with the PacBio platform. They were assembled into 120,926 contigs, and 1185 (82.2%) of the benchmarking universal single-copy orthologs (BUSCO) core genes were completely present in our assembled transcriptome. Furthermore, using 128 Gb of Illumina sequences, the transcript expression was assessed at three stages of Papaver plant development (30, 60, and 90 days), from which we identified 137 differentially expressed transcripts. Furthermore, three co-occurrence heat maps are generated from 51 different plant genomes along with the Papaver transcriptome, i.e., secondary metabolite biosynthesis, isoquinoline alkaloid biosynthesis (BIA) pathway, and cytochrome. Sixty-nine transcripts in the BIA pathway along with 22 different alkaloids (quantified with LC-QTOF-MS/MS) were mapped into the BIA KEGG map (map00950). Finally, we identified 39 full-length cytochrome transcripts and compared them with other genomes. Collectively, this transcriptome data, along with the expression and quantitative metabolite profiles, provides an initial recording of secondary metabolites and their expression related to Papaver plant development. Moreover, these profiles could help to further detail the functional characterization of the various secondary metabolite biosynthesis and Papaver plant development associated problems.

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September 22, 2019

Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

The class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea.We cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S-28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I – III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II?+?III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms.This altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.

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September 22, 2019

Improving eukaryotic genome annotation using single molecule mRNA sequencing.

The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq.We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features.Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies.

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September 22, 2019

Identification and analysis of glutathione S-transferase gene family in sweet potato reveal divergent GST-mediated networks in aboveground and underground tissues in response to abiotic stresses.

Sweet potato, a hexaploid species lacking a reference genome, is one of the most important crops in many developing countries, where abiotic stresses are a primary cause of reduction of crop yield. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in oxidative stress tolerance and cellular detoxification.A total of 42 putative full-length GST genes were identified from two local transcriptome databases and validated by molecular cloning and Sanger sequencing. Sequence and intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into eight subfamilies. Interspecific phylogenetic and comparative analyses indicated that most examined GST paralogs might originate and diverge before the speciation of sweet potato. Results from large-scale RNA-seq and quantitative real-time PCR experiments exhibited extensive variation in gene-expression profiles across different tissues and varieties, which implied strong evolutionary divergence in their gene-expression regulation. Moreover, we performed five manipulated stress experiments and uncovered highly divergent stress-response patterns of sweet potato GST genes in aboveground and underground tissues.Our study identified a large number of sweet potato GST genes, systematically investigated their evolutionary diversification, and provides new insights into the GST-mediated stress-response mechanisms in this worldwide crop.

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September 22, 2019

Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing.

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover.In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts.We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.

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September 22, 2019

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay’s health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.

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September 22, 2019

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.

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September 22, 2019

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

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September 22, 2019

Effect of dietary interventions on the intestinal microbiota of Mongolian hosts

The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairy-oriented daily diets and unique genotype. The aim of the present study was to investigate the effect of switching from the typical high protein and fat Mongolian diets to carbohydrate-rich meals composed principally of wheat, rice and naked oats on the host gut microbiota within 3 weeks. Our study took the advantage of the long sequence reads produced by the PacBio single molecule real-time sequencing technology to enable the profiling of subjects’ gut microbiota communities along the diet intervention to the species precision. We found that the bacterial richness and diversity decreased apparently along the diet intervention. During the diet intervention, the gut microbiota composition displayed no significant difference at phylum level (with major phyla of Firmicutes, Bacteroidetes, Tenericutes and Proteobacteria). The relative abundances of some genera such as Bacteroidetes, Faecalibacterium, Roseburia, Alistipes, Streptococcus, and Oscillospira were significantly altered after the diet switching started. Notably, significant changes were also observed in the proportions of the species Bacteroides dorei, Bacteroides fragilis, Bacteroides thetaiotaomicron, Ruminococcus albus, Ruminococcus faecis, Roseburia faecis and Eubacterium ventriosum. These results have demonstrated that diet and host gut microbiota is closely linked.

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September 22, 2019

Bacterial microbiota composition of fermented fruit and vegetable juices (jiaosu) analyzed by single-molecule, real-time (SMRT) sequencing

Commercially manufactured ‘jiaosu’ (fermented fruit and vegetable juices) have gained popularity in Asia recently. Like other fermented products, they have a high microbial diversity and richness. However, no published study has yet described their microbiota composition. Thus, this work aimed to obtain the full-length 16S rRNA profiles of jiaosu using the PacBio single-molecule, real-time sequencing technology. We described the bacterial microbiota of three jiaosu products purchased from Taiwan and Japan. Bacterial sequences from all three samples distributed across seven different phyla, mainly Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Forty-three genera were identified (e.g. Ochrobactrum, Lactobacillus, Mycobacterium, and Acinetobacter). Fifty- five species were identified (e.g. Ochrobactrum lupini, Mycobacterium abscessus, Acinetobacter john- sonii, Lactobacillus paracasei, Lactobacillus delbrueckii, and Petrobacter succinatimandens). No patho- gen sequences were identified within the entire dataset. Moreover, only a low proportion of sequences represented common skin microflora and the food hygiene indicator Escherichia/ Shigella, suggesting overall acceptable sanitary conditions during the manufacturing process.

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September 22, 2019

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi – macaque model systems and summarizing exciting new progress in this area of research.

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