Menu

Asset Tag: Plant and Animal Sciences,

September 22, 2019

First insights into the nature and evolution of antisense transcription in nematodes.

The development of multicellular organisms is coordinated by various gene regulatory mechanisms that ensure correct spatio-temporal patterns of gene expression. Recently, the role of antisense transcription in gene regulation has moved into focus of research. To characterize genome-wide patterns of antisense transcription and to study their evolutionary conservation, we sequenced a strand-specific RNA-seq library of the nematode Pristionchus pacificus.We identified 1112 antisense configurations of which the largest group represents 465 antisense transcripts (ASTs) that are fully embedded in introns of their host genes. We find that most ASTs show homology to protein-coding genes and are overrepresented in proteomic data. Together with the finding, that expression levels of ASTs and host genes are uncorrelated, this indicates that most ASTs in P. pacificus do not represent non-coding RNAs and do not exhibit regulatory functions on their host genes. We studied the evolution of antisense gene pairs across 20 nematode genomes, showing that the majority of pairs is lineage-specific and even the highly conserved vps-4, ddx-27, and sel-2 loci show abundant structural changes including duplications, deletions, intron gains and loss of antisense transcription. In contrast, host genes in general, are remarkably conserved and encode exceptionally long introns leading to unusually large blocks of conserved synteny.Our study has shown that in P. pacificus antisense transcription as such does not define non-coding RNAs but is rather a feature of highly conserved genes with long introns. We hypothesize that the presence of regulatory elements imposes evolutionary constraint on the intron length, but simultaneously, their large size makes them a likely target for translocation of genomic elements including protein-coding genes that eventually end up as ASTs.

Read more
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

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

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

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

Read more
September 22, 2019

Complete genome sequences of two genotype A2 small ruminant lentiviruses isolated from infected U.S. sheep.

Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs) have been identified in the United States that infect sheep with specific host transmembrane protein 154 (TMEM154) diplotypes. Here, we report the first two complete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype A2, subgroups 1 and 2. Copyright © 2017 Workman et al.

Read more
September 22, 2019

Alternative isoform analysis of Ttc8 expression in the rat pineal gland using a multi-platform sequencing approach reveals neural regulation.

Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome.

Read more
September 22, 2019

Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for =200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3′ sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.

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.