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October 23, 2019

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.

Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.

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October 23, 2019

Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications. Copyright © 2017. Published by Elsevier Inc.

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October 23, 2019

Chromosomal-level assembly of yellow catfish genome using third-generation DNA sequencing and Hi-C analysis.

The yellow catfish, Pelteobagrus fulvidraco, belonging to the Siluriformes order, is an economically important freshwater aquaculture fish species in Asia, especially in Southern China. The aquaculture industry has recently been facing tremendous challenges in germplasm degeneration and poor disease resistance. As the yellow catfish exhibits notable sex dimorphism in growth, with adult males about two- to three-fold bigger than females, the way in which the aquaculture industry takes advantage of such sex dimorphism is another challenge. To address these issues, a high-quality reference genome of the yellow catfish would be a very useful resource.To construct a high-quality reference genome for the yellow catfish, we generated 51.2 Gb short reads and 38.9 Gb long reads using Illumina and Pacific Biosciences (PacBio) sequencing platforms, respectively. The sequencing data were assembled into a 732.8 Mb genome assembly with a contig N50 length of 1.1 Mb. Additionally, we applied Hi-C technology to identify contacts among contigs, which were then used to assemble contigs into scaffolds, resulting in a genome assembly with 26 chromosomes and a scaffold N50 length of 25.8 Mb. Using 24,552 protein-coding genes annotated in the yellow catfish genome, the phylogenetic relationships of the yellow catfish with other teleosts showed that yellow catfish separated from the common ancestor of channel catfish ~81.9 million years ago. We identified 1,717 gene families to be expanded in the yellow catfish, and those gene families are mainly enriched in the immune system, signal transduction, glycosphingolipid biosynthesis, and fatty acid biosynthesis.Taking advantage of Illumina, PacBio, and Hi-C technologies, we constructed the first high-quality chromosome-level genome assembly for the yellow catfish P. fulvidraco. The genomic resources generated in this work not only offer a valuable reference genome for functional genomics studies of yellow catfish to decipher the economic traits and sex determination but also provide important chromosome information for genome comparisons in the wider evolutionary research community.

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September 22, 2019

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

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September 22, 2019

TACO produces robust multisample transcriptome assemblies from RNA-seq.

Accurate transcript structure and abundance inference from RNA sequencing (RNA-seq) data is foundational for molecular discovery. Here we present TACO, a computational method to reconstruct a consensus transcriptome from multiple RNA-seq data sets. TACO employs novel change-point detection to demarcate transcript start and end sites, leading to improved reconstruction accuracy compared with other tools in its class. The tool is available at http://tacorna.github.io and can be readily incorporated into RNA-seq analysis workflows.

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September 22, 2019

Membrane attack complex-associated molecules from redlip mullet (Liza haematocheila): Molecular characterization and transcriptional evidence of C6, C7, C8ß, and C9 in innate immunity.

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8ß and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3′ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

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September 22, 2019

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

neoantigenR: An annotation based pipeline for tumor neoantigen identification from sequencing data

Studies indicate that more than 90% of human genes are alternatively spliced, suggesting the complexity of the transcriptome assembly and analysis. The splicing process is often disrupted, resulting in both functional and non-functional end-products (Sveen et al. 2016) in many cancers. Harnessing the immune system to fight against malignant cancers carrying aberrantly mutated or spliced products is becoming a promising approach to cancer therapy. Advances in immune checkpoint blockade have elicited adaptive immune responses with promising clinical responses to treatments against human malignancies (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Emerging data suggest that recognition of patient-specific mutation-associated cancer antigens (i.e. from alternative splicing isoforms) may allow scientists to dissect the immune response in the activity of clinical immunotherapies (Schumacher and Schreiber 2015). The advent of high-throughput sequencing technology has provided a comprehensive view of both splicing aberrations and somatic mutations across a range of human malignancies, allowing for a deeper understanding of the interplay of various disease mechanisms. Meanwhile, studies show that the number of transcript isoforms reported to date may be limited by the short-read sequencing due to the inherit limitation of transcriptome reconstruction algorithms, whereas long-read sequencing is able to significantly improve the detection of alternative splicing variants since there is no need to assemble full-length transcripts from short reads. The analysis of these high-throughput long-read sequencing data may permit a systematic view of tumor specific peptide epitopes (also known as neoantigens) that could serve as targets for immunotherapy (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Currently, there is no software pipeline available that can efficiently produce mutation-associated cancer antigens from raw high-throughput sequencing data on patient tumor DNA (The Problem with Neoantigen Prediction 2017). In addressing this issue, we introduce a R package that allows the discoveries of peptide epitope candidates, which are the tumor-specific peptide fragments containing potential functional neoantigens. These peptide epitopes consist of structure variants including insertion, deletions, alternative sequences, and peptides from nonsynonymous mutations. Analysis of these precursor candidates with widely used tools such as netMHC allows for the accurate in-silico prediction of neoantigens. The pipeline named neoantigeR is currently hosted in https://github.com/ICBI/neoantigeR.

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September 22, 2019

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019

IsoSeq analysis and functional annotation of the infratentorial ependymoma tumor tissue on PacBio RSII platform.

Here, we sequenced and functionally annotated the long reads (1-2 kb) cDNAs library of an infratentorial ependymoma tumor tissue on PacBio RSII by Iso-Seq protocol using SMRT technology. 577 MB, data was generated from the brain tissues of ependymoma tumor patient, producing 1,19,313 high-quality reads assembled into 19,878 contigs using Celera assembler followed by Quiver pipelines, which produced 2952 unique protein accessions in the nr protein database and 307 KEGG pathways. Additionally, when we compared GO terms of second and third level with alternative splicing data obtained through HTA Array2.0. We identified four and twelve transcript cluster IDs in Level-2 and Level-3 scores respectively with alternative splicing index predicting mainly the major pathways of hallmarks of cancer. Out of these transcript cluster IDs only transcript cluster IDs of gene PNMT, SNN and LAMB1 showed Reads Per Kilobase of exon model per Million mapped reads (RPKM) values at gene-level expression (GE) and transcript-level (TE) track. Most importantly, brain-specific genes–PNMT, SNN and LAMB1 show their involvement in Ependymoma.

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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

16S rRNA long-read sequencing of the granulation tissue from nonsmokers and smokers-severe chronic periodontitis patients

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

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September 22, 2019

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing.

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1(89.6) produces at least 109 different spliced RNAs, including a previously unappreciated ~1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.

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