Menu

Asset Tag: PacBio

September 22, 2019

Transcriptome comparative analysis of salt stress responsiveness in chrysanthemum (Dendranthema grandiflorum) roots by Illumina- and Single-Molecule Real-Time-based RNA sequencing.

Salt response has long been considered a polygenic-controlled character in plants. Under salt stress conditions, plants respond by activating a great amount of proteins and enzymes. To develop a better understanding of the molecular mechanism and screen salt responsive genes in chrysanthemum under salt stress, we performed the RNA sequencing (RNA-seq) on both salt-processed chrysanthemum seedling roots and the control group, and gathered six cDNA databases eventually. Moreover, to overcome the Illumina HiSeq technology’s limitation on sufficient length of reads and improve the quality and accuracy of the result, we combined Illumina HiSeq with single-molecule real-time sequencing (SMRT-seq) to decode the full-length transcripts. As a result, we successfully collected 550,823 unigenes, and from which we selected 48,396 differentially expressed genes (DEGs). Many of these DEGs were associated with the signal transduction, biofilm system, antioxidant system, and osmotic regulation system, such as mitogen-activated protein kinase (MAPK), Acyl-CoA thioesterase (ACOT), superoxide (SOD), catalase (CAT), peroxisomal membrane protein (PMP), and pyrroline-5-carboxylate reductase (P5CR). The quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 15 unigenes was performed to test the data validity. The results were highly consistent with the RNA-seq results. In all, these findings could facilitate further detection of the responsive molecular mechanism under salt stress. They also provided more accurate candidate genes for genetic engineering on salt-tolerant chrysanthemums.

Read more
September 22, 2019

Microsatellites from Fosterella christophii (Bromeliaceae) by de novo transcriptome sequencing on the Pacific Biosciences RS platform.

Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula.Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively.The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.

Read more
September 22, 2019

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These “phasiRNAs” are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.© 2018 Kakrana et al.; Published by Cold Spring Harbor Laboratory Press.

Read more
September 22, 2019

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

Read more
September 22, 2019

Melanization of mycorrhizal fungal necromass structures microbial decomposer communities

Mycorrhizal fungal necromass is increasingly recognized as an important contributor to soil organic carbon pools, particularly in forest ecosystems. While its decomposition rate is primarily determined by biochemical composition, how traits such as melanin content affect the structure of necromass decomposer communities remains poorly understood. To assess the role of biochemical traits on microbial decomposer community composition and functioning, we incubated melanized and non-melanized necromass of the mycorrhizal fungus Meliniomyces bicolor in Pinus- and Quercus-dominated forests in Minnesota, USA and then assessed the associated fungal and bacterial decomposer communities after 1, 2 and 3 months using high-throughput sequencing. Melanized necromass decomposed significantly slower than non-melanized necromass in both forests. The structure of the microbial decomposer communities depended significantly on necromass melanin content, although the effect was stronger for fungi than bacteria. On non-melanized necromass, fungal communities were dominated by r-selected ascomycete and mucoromycete microfungi early and then replaced by basidiomycete ectomycorrhizal fungi, while on melanized necromass these groups were co-dominant throughout the incubation. Bacterial communities were dominated by both specialist mycophageous and generalist taxa. Synthesis. Our results indicate that necromass biochemistry not only strongly affects rates of decomposition but also the structure of the associated decomposer communities. Furthermore, the observed colonization patterns suggest that fungi, and particularly ectomycorrhizal fungi, may play a more important role in necromass decomposition than previously recognized.

Read more
September 22, 2019

Cow-to-mouse fecal transplantations suggest intestinal microbiome as one cause of mastitis.

Mastitis, which affects nearly all lactating mammals including human, is generally thought to be caused by local infection of the mammary glands. For treatment, antibiotics are commonly prescribed, which however are of concern in both treatment efficacy and neonate safety. Here, using bovine mastitis which is the most costly disease in the dairy industry as a model, we showed that intestinal microbiota alone can lead to mastitis.Fecal microbiota transplantation (FMT) from mastitis, but not healthy cows, to germ-free (GF) mice resulted in mastitis symptoms in mammary gland and inflammations in serum, spleen, and colon. Probiotic intake in parallel with FMT from diseased cows led to relieved mastitis symptoms in mice, by shifting the murine intestinal microbiota to a state that is functionally distinct from either healthy or diseased microbiota yet structurally similar to the latter. Despite conservation in mastitis symptoms, diseased cows and mice shared few mastitis-associated bacterial organismal or functional markers, suggesting striking divergence in mastitis-associated intestinal microbiota among lactating mammals. Moreover, an “amplification effect” of disease-health distinction in both microbiota structure and function was apparent during the cow-to-mouse FMT.Hence, dysbiosis of intestinal microbiota may be one cause of mastitis, and probiotics that restore intestinal microbiota function are an effective and safe strategy to treat mastitis.

Read more
September 22, 2019

Long-read sequencing of chicken transcripts and identification of new transcript isoforms.

The chicken has long served as an important model organism in many fields, and continues to aid our understanding of animal development. Functional genomics studies aimed at probing the mechanisms that regulate development require high-quality genomes and transcript annotations. The quality of these resources has improved dramatically over the last several years, but many isoforms and genes have yet to be identified. We hope to contribute to the process of improving these resources with the data presented here: a set of long cDNA sequencing reads, and a curated set of new genes and transcript isoforms not currently represented in the most up-to-date genome annotation currently available to the community of researchers who rely on the chicken genome.

Read more
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.

Read more
September 22, 2019

Isoform sequencing provides insight into natural genetic diversity in maize.

W64A, as a member of non-stiff stalk maize, has been used to develop current corn in plant breeding, and serving as one of broadest parent line for the commercial hybrid seed production (Huffman, 1984). The inbred had the characteristics of early flowering, average plant and ear height at its maturity, very strong roots and good stalks (Runge, 2004). In addition, W64A serves as an invaluable germplasm to study gene functions especially in the field of corn nutrition and endosperm texture given its nearly complete vitreousness and hardness (Figure 1a). However, little is known about the background of genetic and genomic information for W64A. With the advent of the revolutionary technology of PacBio long-read sequencing, we can simultaneously obtain a large amount of full-length cDNA up to 20 kb (An et al., 2018). This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

Read more
September 22, 2019

Evaluation of bacterial contamination in raw milk, ultra-high temperature milk and infant formula using single molecule, real-time sequencing technology.

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Read more
September 22, 2019

Quantitative metaproteomics highlight the metabolic contributions of uncultured phylotypes in a thermophilic anaerobic digester.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH3-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum “Atribacteria” These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ß-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor.Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ß-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane. Copyright © 2016 American Society for Microbiology.

Read more
September 22, 2019

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested.Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes.MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Read more
September 22, 2019

A genomic case study of mixed fibrolamellar hepatocellular carcinoma.

Mixed fibrolamellar hepatocellular carcinoma (mFL-HCC) is a rare liver tumor defined by the presence of both pure FL-HCC and conventional HCC components, represents up to 25% of cases of FL-HCC, and has been associated with worse prognosis. Recent genomic characterization of pure FL-HCC identified a highly recurrent transcript fusion (DNAJB1:PRKACA) not found in conventional HCC.We performed exome and transcriptome sequencing of a case of mFL-HCC. A novel BAC-capture approach was developed to identify a 400 kb deletion as the underlying genomic mechanism for a DNAJB1:PRKACA fusion in this case. A sensitive Nanostring Elements assay was used to screen for this transcript fusion in a second case of mFL-HCC, 112 additional HCC samples and 44 adjacent non-tumor liver samples.We report the first comprehensive genomic analysis of a case of mFL-HCC. No common HCC-associated mutations were identified. The very low mutation rate of this case, large number of mostly single-copy, long-range copy number variants, and high expression of ERBB2 were more consistent with previous reports of pure FL-HCC than conventional HCC. In particular, the DNAJB1:PRKACA fusion transcript specifically associated with pure FL-HCC was detected at very high expression levels. Subsequent analysis revealed the presence of this fusion in all primary and metastatic samples, including those with mixed or conventional HCC pathology. A second case of mFL-HCC confirmed our finding that the fusion was detectable in conventional components. An expanded screen identified a third case of fusion-positive HCC, which upon review, also had both conventional and fibrolamellar features. This screen confirmed the absence of the fusion in all conventional HCC and adjacent non-tumor liver samples.These results indicate that mFL-HCC is similar to pure FL-HCC at the genomic level and the DNAJB1:PRKACA fusion can be used as a diagnostic tool for both pure and mFL-HCC.© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.