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Asset Tag: PacBio RS II

September 22, 2019

Impacts of experimentally accelerated forest succession on belowground plant and fungal communities

Understanding how soil processes, belowground plant and fungal species composition, and nutrient cycles are altered by disturbances is essential for understanding the role forests play in mitigating global climate change. Here we ask: How are root and fungal communities altered in a mid-successional forest during shifts in dominant tree species composition? This study utilizes the Forest Accelerated Succession ExperimenT (FASET) at the University of Michigan Biological Station (UMBS) as a platform for addressing this question. FASET consists of a 39-ha treatment in which all mature early successional aspen (Populus spp.) and paper birch (Betula papyrifera) were killed by stem-girdling in 2008. Four years after girdling, neither overall fungal diversity indices, plant diversity indices, nor root biomass differed between girdled (treated) and non-girdled (reference) stands. However, experimental advancement of succession by removal of aspen and birch resulted in 1) a shift in fungal functional groups, with significantly less ectomycorrhizal fungi, 2) a trend toward less arbuscular mycorrhizal fungi, and 3) a significant increase in the proportion of saprotrophs in girdled stands. In addition to shifts in functional groups between treated and untreated stands, ectomycorrhizal fungi proportions were negatively correlated with NH4+ and total dissolved inorganic nitrogen (DIN) in soil. This research illustrates the propensity for disturbances in forest ecosystems to shift fungal community composition, which has implications for carbon storage and nutrient cycling in soils under future climate scenarios.

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September 22, 2019

Characterization of novel transcripts in pseudorabies virus.

In this study we identified two 3′-coterminal RNA molecules in the pseudorabies virus. The highly abundant short transcript (CTO-S) proved to be encoded between the ul21 and ul22 genes in close vicinity of the replication origin (OriL) of the virus. The less abundant long RNA molecule (CTO-L) is a transcriptional readthrough product of the ul21 gene and overlaps OriL. These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system. It emerged that transcription of the CTOs is fully dependent on the viral transactivator protein IE180 and CTO-S is not a microRNA precursor. We propose an interaction between the transcription and replication machineries at this genomic location, which might play an important role in the regulation of DNA synthesis.

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September 22, 2019

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides.

Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness. Copyright © 2015, American Association for the Advancement of Science.

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September 22, 2019

Single-Molecule Long-Read Sequencing of Zanthoxylum bungeanum Maxim. Transcriptome: Identification of Aroma-Related Genes

Zanthoxylum bungeanum Maxim. is an economically important tree species that is resistant to drought and infertility, and has potential medicinal and edible value. However, comprehensive genomic data are not yet available for this species, limiting its potential utility for medicinal use, breeding programs, and cultivation. Transcriptome sequencing provides an effective approach to remedying this shortcoming. Herein, single-molecule long-read sequencing and next-generation sequencingapproacheswereusedinparalleltoobtaintranscriptisoformstructureandgenefunctional informationinZ.bungeanum. Intotal, 282,101readsofinserts(ROIs)wereidentified, including134,074 full-length non-chimeric reads, among which 65,711 open reading frames (ORFs), 50,135 simple sequence repeats (SSRs), and 1492 long non-coding RNAs (lncRNAs) were detected. Functional annotation revealed metabolic pathways related to aroma components and color characteristics in Z. bungeanum. Unexpectedly, 30 transcripts were annotated as genes involved in regulating the pathogenesis of breast and colorectal cancers. This work provides a comprehensive transcriptome resource for Z. bungeanum, and lays a foundation for the further investigation and utilization of Zanthoxylum resources.

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September 22, 2019

Extensive alternative splicing of KIR transcripts.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.

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September 22, 2019

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq.Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset.The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.

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September 22, 2019

Full-length transcriptome survey and expression analysis of Cassia obtusifolia to discover putative genes related to aurantio-obtusin biosynthesis, seed formation and development, and stress response.

The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation and development, and stress response as well in C. obtusifolia.

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September 22, 2019

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

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September 22, 2019

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.

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September 22, 2019

PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme.

High-throughput transcriptome sequencing (RNA-seq) technology promises to discover novel protein-coding and non-coding transcripts, particularly the identification of long non-coding RNAs (lncRNAs) from de novo sequencing data. This requires tools that are not restricted by prior gene annotations, genomic sequences and high-quality sequencing.We present an alignment-free tool called PLEK (predictor of long non-coding RNAs and messenger RNAs based on an improved k-mer scheme), which uses a computational pipeline based on an improved k-mer scheme and a support vector machine (SVM) algorithm to distinguish lncRNAs from messenger RNAs (mRNAs), in the absence of genomic sequences or annotations. The performance of PLEK was evaluated on well-annotated mRNA and lncRNA transcripts. 10-fold cross-validation tests on human RefSeq mRNAs and GENCODE lncRNAs indicated that our tool could achieve accuracy of up to 95.6%. We demonstrated the utility of PLEK on transcripts from other vertebrates using the model built from human datasets. PLEK attained >90% accuracy on most of these datasets. PLEK also performed well using a simulated dataset and two real de novo assembled transcriptome datasets (sequenced by PacBio and 454 platforms) with relatively high indel sequencing errors. In addition, PLEK is approximately eightfold faster than a newly developed alignment-free tool, named Coding-Non-Coding Index (CNCI), and 244 times faster than the most popular alignment-based tool, Coding Potential Calculator (CPC), in a single-threading running manner.PLEK is an efficient alignment-free computational tool to distinguish lncRNAs from mRNAs in RNA-seq transcriptomes of species lacking reference genomes. PLEK is especially suitable for PacBio or 454 sequencing data and large-scale transcriptome data. Its open-source software can be freely downloaded from https://sourceforge.net/projects/plek/files/.

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September 22, 2019

Long-read, Single Molecule, Real-Time (SMRT) DNA Sequencing for metagenomic applications

In this chapter, we describe applications of single molecule, real-time (SMRT) DNA sequencing toward metagenomic research. The long sequence reads, combined with a lack of bias with respect to DNA sequence context or GC content, facilitate a more comprehensive analysis of the genomic constitution of microbial communities. Full-length 16S RNA gene sequencing at high (>99%) accuracy allows for species-level characterization of community members concomitant with the determination of community structure. The application of SMRT sequencing to whole-community shotgun microbial metagenomics has also been discussed.

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September 22, 2019

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

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September 22, 2019

Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession.

Although we understand the ecological processes eliciting changes in plant community composition during secondary succession, we do not understand whether co-occurring changes in plant detritus shape saprotrophic microbial communities in soil. In this study, we investigated soil microbial composition and function across an old-field chronosequence ranging from 16 to 86 years following agricultural abandonment, as well as three forests representing potential late-successional ecosystems. Fungal and bacterial community composition was quantified from ribosomal DNA, and insight into the functional potential of the microbial community to decay plant litter was gained from shotgun metagenomics and extracellular enzyme assays. Accumulation of soil organic matter across the chronosequence exerted a positive and significant effect on fungal phylogenetic ß-diversity and the activity of extracellular enzymes with lignocellulolytic activity. In addition, the increasing abundance of lignin-rich C4 grasses was positively related to the composition of fungal genes with lignocellulolytic function, thereby linking plant community composition, litter biochemistry, and microbial community function. However, edaphic properties were the primary agent shaping bacterial communities, as bacterial ß-diversity and variation in functional gene composition displayed a significant and positive relationship to soil pH across the chronosequence. The late-successional forests were compositionally distinct from the oldest old fields, indicating that substantial changes occur in soil microbial communities as old fields give way to forests. Taken together, our observations demonstrate that plants govern the turnover of soil fungal communities and functional characteristics during secondary succession, due to the continual input of detritus and differences in litter biochemistry among plant species.

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September 22, 2019

Single Molecule Sequencing: new outlooks for solving genome assembly and transcripts identification challenges

In this review, we introduce a novel sequencing technology, named Single Molecule Real Time sequencing. Also called Single Molecule Sequencing, as it do not requires any amplification, this new technology is able to pro- duce much longer reads than previous NGS technologies such as Illumina. This read size improvements, which can reach 150 fold, will solve many challenges caused by the actual NGS technologies. Short NGS reads, reach- ing a maximum size of 300 bp, make it hard to reconstitute a whole genome and are always leading to fragmented genome assembly. It is also difficult to correctly infer transcript quantification and identification when there is a high isoforms diversity. Despite their higher error rate, long reads have shown very promising result concerning these actual issues. We show that longer reads can produce less fragmented assembly, with a better quality, but also sequence from start to end mRNA, making it much more easier to infer correct transcript quantification, and even allow new intron structure and so new isoforms discovery.

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September 22, 2019

Characterization of the Rosellinia necatrix transcriptome and genes related to pathogenesis by single-molecule mRNA sequencing.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world’s most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

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