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September 22, 2019  |  

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

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September 22, 2019  |  

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing such granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.

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September 22, 2019  |  

Carbohydrate staple food modulates gut microbiota of Mongolians in China.

Gut microbiota is a determining factor in human physiological functions and health. It is commonly accepted that diet has a major influence on the gut microbial community, however, the effects of diet is not fully understood. The typical Mongolian diet is characterized by high and frequent consumption of fermented dairy products and red meat, and low level of carbohydrates. In this study, the gut microbiota profile of 26 Mongolians whom consumed wheat, rice and oat as the sole carbohydrate staple food for a week each consecutively was determined. It was observed that changes in staple carbohydrate rapidly (within a week) altered gut microbial community structure and metabolic pathway of the subjects. Wheat and oat favored bifidobacteria (Bifidobacterium catenulatum, Bifodobacteriumbifidum, Bifidobacterium adolescentis); whereas rice suppressed bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) and wheat suppresses Lactobaciilus, Ruminococcus and Bacteroides. The study exhibited two gut microbial clustering patterns with the preference of fucosyllactose utilization linking to fucosidase genes (glycoside hydrolase family classifications: GH95 and GH29) encoded by Bifidobacterium, and xylan and arabinoxylan utilization linking to xylanase and arabinoxylanase genes encoded by Bacteroides. There was also a correlation between Lactobacillus ruminis and sialidase, as well as Butyrivibrio crossotus and xylanase/xylosidase. Meanwhile, a strong concordance was found between the gastrointestinal bacterial microbiome and the intestinal virome. Present research will contribute to understanding the impacts of the dietary carbohydrate on human gut microbiome, which will ultimately help understand relationships between dietary factor, microbial populations, and the health of global humans.

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September 22, 2019  |  

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019  |  

Defining a personal, allele-specific, and single-molecule long-read transcriptome.

Personal transcriptomes in which all of an individual’s genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes =3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV–in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome.

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September 22, 2019  |  

Molecular characterization of eukaryotic algal communities in the tropical phyllosphere based on real-time sequencing of the 18S rDNA gene.

Foliicolous algae are a common occurrence in tropical forests. They are referable to a few simple morphotypes (unicellular, sarcinoid-like or filamentous), which makes their morphology of limited usefulness for taxonomic studies and species diversity assessments. The relationship between algal community and their host phyllosphere was not clear. In order to obtain a more accurate assessment, we used single molecule real-time sequencing of the 18S rDNA gene to characterize the eukaryotic algal community in an area of South-western China.We annotated 2922 OTUs belonging to five classes, Ulvophyceae, Trebouxiophyceae, Chlorophyceae, Dinophyceae and Eustigmatophyceae. Novel clades formed by large numbers sequences of green algae were detected in the order Trentepohliales (Ulvophyceae) and the Watanabea clade (Trebouxiophyceae), suggesting that these foliicolous communities may be substantially more diverse than so far appreciated and require further research. Species in Trentepohliales, Watanabea clade and Apatococcus clade were detected as the core members in the phyllosphere community studied. Communities from different host trees and sampling sites were not significantly different in terms of OTUs composition. However, the communities of Musa and Ravenala differed from other host plants significantly at the genus level, since they were dominated by Trebouxiophycean epiphytes.The cryptic diversity of eukaryotic algae especially Chlorophytes in tropical phyllosphere is very high. The community structure at species-level has no significant relationship either with host phyllosphere or locations. The core algal community in tropical phyllopshere is consisted of members from Trentepohliales, Watanabea clade and Apatococcus clade. Our study provided a large amount of novel 18S rDNA sequences that will be useful to unravel the cryptic diversity of phyllosphere eukaryotic algae and for comparisons with similar future studies on this type of communities.

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September 22, 2019  |  

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform.The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated.We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.

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September 22, 2019  |  

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens.Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity.Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

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September 22, 2019  |  

Construction of Pará rubber tree genome and multi-transcriptome database accelerates rubber researches.

Natural rubber is an economically important material. Currently the Pará rubber tree, Hevea brasiliensis is the main commercial source. Little is known about rubber biosynthesis at the molecular level. Next-generation sequencing (NGS) technologies brought draft genomes of three rubber cultivars and a variety of RNA sequencing (RNA-seq) data. However, no current genome or transcriptome databases (DB) are organized by gene.A gene-oriented database is a valuable support for rubber research. Based on our original draft genome sequence of H. brasiliensis RRIM600, we constructed a rubber tree genome and transcriptome DB. Our DB provides genome information including gene functional annotations and multi-transcriptome data of RNA-seq, full-length cDNAs including PacBio Isoform sequencing (Iso-Seq), ESTs and genome wide transcription start sites (TSSs) derived from CAGE technology. Using our original and publically available RNA-seq data, we calculated co-expressed genes for identifying functionally related gene sets and/or genes regulated by the same transcription factor (TF). Users can access multi-transcriptome data through both a gene-oriented web page and a genome browser. For the gene searching system, we provide keyword search, sequence homology search and gene expression search; users can also select their expression threshold easily.The rubber genome and transcriptome DB provides rubber tree genome sequence and multi-transcriptomics data. This DB is useful for comprehensive understanding of the rubber transcriptome. This will assist both industrial and academic researchers for rubber and economically important close relatives such as R. communis, M. esculenta and J. curcas. The Rubber Transcriptome DB release 2017.03 is accessible at http://matsui-lab.riken.jp/rubber/ .

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September 22, 2019  |  

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei

Despite the large interest in the human microbiome in recent years, there are no reports of bacterial DNA methylation in the microbiome. Here metagenomic sequencing using the Pacific Biosciences platform allowed for rapid identification of bacterial GATC methylation status of a bacterial species in human stool samples. For this work, two stool samples were chosen that were dominated by a single species, Bacteroides dorei. Based on 16S rRNA analysis, this species represented over 45% of the bacteria present in these two samples. The B. dorei genome sequence from these samples was determined and the GATC methylation sites mapped. The Bacteroides dorei genome from one subject lacked any GATC methylation and lacked the DNA adenine methyltransferase genes. In contrast, B. dorei from another subject contained 20,551 methylated GATC sites. Of the 4970 open reading frames identified in the GATC methylated B. dorei genome, 3184 genes were methylated as well as 1735 GATC methylations in intergenic regions. These results suggest that DNA methylation patterns are important to consider in multi-omic analyses of microbiome samples seeking to discover the diversity of bacterial functions and may differ between disease states.

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September 22, 2019  |  

Global dissection of alternative splicing uncovers transcriptional diversity in tissues and associates with the flavonoid pathway in tea plant (Camellia sinensis).

Alternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea.In our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~?20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc. Thirteen co-expressed modules of AS transcripts were identified, which revealed a similar pattern between the bud and young leaves as well as a distinct pattern between seasons. AS events of structural genes including anthocyanidin reductase and MYB transcription factors were involved in biosynthesis of flavonoid, especially in vegetative tissues. The AS isoforms rather than the full-length ones were the major transcripts involved in flavonoid synthesis pathway, and is positively correlated with the catechins content conferring the tea taste. We propose that the AS is an important functional mechanism in regulating flavonoid metabolites.Our study provides the insight into the AS events underlying tea plant’s uniquely different developmental process and highlights the important contribution and efficacy of alternative splicing regulatory function to biosynthesis of flavonoids.

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September 22, 2019  |  

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm.

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.

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September 22, 2019  |  

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

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September 22, 2019  |  

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781?bp per gene in 82.473?Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals. Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019  |  

Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts.

Polyploidization contributes to the complexity of gene expression, resulting in numerous related but different transcripts. This study explored the transcriptome diversity and complexity of the tetraploid Arabica coffee (Coffea arabica) bean. Long-read sequencing (LRS) by Pacbio Isoform sequencing (Iso-seq) was used to obtain full-length transcripts without the difficulty and uncertainty of assembly required for reads from short-read technologies. The tetraploid transcriptome was annotated and compared with data from the sub-genome progenitors. Caffeine and sucrose genes were targeted for case analysis. An isoform-level tetraploid coffee bean reference transcriptome with 95 995 distinct transcripts (average 3236 bp) was obtained. A total of 88 715 sequences (92.42%) were annotated with BLASTx against NCBI non-redundant plant proteins, including 34 719 high-quality annotations. Further BLASTn analysis against NCBI non-redundant nucleotide sequences, Coffea canephora coding sequences with UTR, C. arabica ESTs, and Rfam resulted in 1213 sequences without hits, were potential novel genes in coffee. Longer UTRs were captured, especially in the 5?UTRs, facilitating the identification of upstream open reading frames. The LRS also revealed more and longer transcript variants in key caffeine and sucrose metabolism genes from this polyploid genome. Long sequences (>10 kilo base) were poorly annotated. LRS technology shows the limitation of previous studies. It provides an important tool to produce a reference transcriptome including more of the diversity of full-length transcripts to help understand the biology and support the genetic improvement of polyploid species such as coffee.© The Authors 2017. Published by Oxford University Press.

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