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September 22, 2019  |  

HapIso: An accurate method for the haplotype-specific isoforms reconstruction from long single-molecule reads

Sequencing of RNA provides the possibility to study an individual’s transcriptome landscape and determine allelic expression ratios. Single-molecule protocols generate multi-kilobase reads longer than most transcripts allowing sequencing of complete haplotype isoforms. This allows partitioning the reads into two parental haplotypes. While the read length of the single-molecule protocols is long, the relatively high error rate limits the ability to accurately detect the genetic variants and assemble them into the haplotype-specific isoforms. In this paper, we present HapIso (Haplotype-specific Isoform Reconstruction), a method able to tolerate the relatively high error-rate of the single-molecule platform and partition the isoform reads into the parental alleles. Phasing the reads according to the allele of origin allows our method to efficiently distinguish between the read errors and the true biological mutations. HapIso uses a k-means clustering algorithm aiming to group the reads into two meaningful clusters maximizing the similarity of the reads within cluster and minimizing the similarity of the reads from different clusters. Each cluster corresponds to a parental haplotype. We use family pedigree information to evaluate our approach. Experimental validation suggests that HapIso is able to tolerate the relatively high error-rate and accurately partition the reads into the parental alleles of the isoform transcripts. Furthermore, our method is the first method able to reconstruct the haplotype-specific isoforms from long single-molecule reads. The open source Python implementation of HapIso is freely available for download at https://?github.?com/?smangul1/?HapIso/?.

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September 22, 2019  |  

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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September 22, 2019  |  

SuperTranscripts: a data driven reference for analysis and visualisation of transcriptomes.

Numerous methods have been developed to analyse RNA sequencing (RNA-seq) data, but most rely on the availability of a reference genome, making them unsuitable for non-model organisms. Here we present superTranscripts, a substitute for a reference genome, where each gene with multiple transcripts is represented by a single sequence. The Lace software is provided to construct superTranscripts from any set of transcripts, including de novo assemblies. We demonstrate how superTranscripts enable visualisation, variant detection and differential isoform detection in non-model organisms. We further use Lace to combine reference and assembled transcriptomes for chicken and recover hundreds of gaps in the reference genome.

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September 22, 2019  |  

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

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September 22, 2019  |  

Direct chromosome-length haplotyping by single-cell sequencing.

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid genomes along the entire length of all chromosomes. We demonstrate this by building a complete haplotype for a HapMap individual (NA12878) at high accuracy (concordance 99.3%), without using generational information or statistical inference. By use of this approach, we mapped all meiotic recombination events in a family trio with high resolution (median range ~14 kb) and phased larger structural variants like deletions, indels, and balanced rearrangements like inversions. Lastly, the single-cell resolution of Strand-seq allowed us to observe loss of heterozygosity regions in a small number of cells, a significant advantage for studies of heterogeneous cell populations, such as cancer cells. We conclude that Strand-seq is a unique and powerful approach to completely phase individual genomes and map inheritance patterns in families, while preserving haplotype differences between single cells.© 2016 Porubský et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Emergence, retention and selection: A trilogy of origination for functional de novo proteins from ancestral lncRNAs in primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

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September 22, 2019  |  

Minimap2: pairwise alignment for nucleotide sequences.

Recent advances in sequencing technologies promise ultra-long reads of ~100 kb in average, full-length mRNA or cDNA reads in high throughput and genomic contigs over 100 Mb in length. Existing alignment programs are unable or inefficient to process such data at scale, which presses for the development of new alignment algorithms.Minimap2 is a general-purpose alignment program to map DNA or long mRNA sequences against a large reference database. It works with accurate short reads of =100?bp in length, =1?kb genomic reads at error rate ~15%, full-length noisy Direct RNA or cDNA reads and assembly contigs or closely related full chromosomes of hundreds of megabases in length. Minimap2 does split-read alignment, employs concave gap cost for long insertions and deletions and introduces new heuristics to reduce spurious alignments. It is 3-4 times as fast as mainstream short-read mappers at comparable accuracy, and is =30 times faster than long-read genomic or cDNA mappers at higher accuracy, surpassing most aligners specialized in one type of alignment.https://github.com/lh3/minimap2.Supplementary data are available at Bioinformatics online.

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September 22, 2019  |  

Improved high-quality genome assembly and annotation of Tibetan hulless barley

Background The Tibetan hulless barley (Hordeum vulgare L. var. nudum), also called textquotedblleftQingketextquotedblright in Chinese and textquotedblleftNetextquotedblright in Tibetan, is the staple food for Tibetans and an important livestock feed in the Tibetan Plateau. The Tibetan hulless barley in China has about 3500 years of cultivation history, mainly produced in Tibet, Qinghai, Sichuan, Yunnan and other areas. In addition, Tibetan hulless barley has rich nutritional value and outstanding health effects, including the beta glucan, dietary fiber, amylopectin, the contents of trace elements, which are higher than any other cereal crops.Findings Here, we reported an improved high-quality assembly of Tibetan hulless barley genome with 4.0 Gb in size. We employed the falcon assembly package, scaffolding and error correction tools to finish improvement using PacBio long reads sequencing technology, with contig and scaffold N50 lengths of 1.563Mb and 4.006Mb, respectively, representing more continuous than the original Tibetan hulless barley genome nearly two orders of magnitude. We also re-annotated the new assembly, and reported 61,303 stringent confident putative protein-coding genes, of which 40,457 is HC genes. We have developed a new Tibetan hulless barley genome database (THBGD) to download and use friendly, as well as to better manage the information of the Tibetan hulless barley genetic resources.Conclusions The availability of new Tibetan hulless barley genome and annotations will take the genetics of Tibetan hulless barley to a new level and will greatly simplify the breeders effort. It will also enrich the granary of the Tibetan people.AbbreviationsBLASTBasic Local Alignment Search ToolBUSCOBenchmarking Universal Single-Copy OrthologsQVquality valuePacBioPacifc BiosciencesRNA-seqRNA sequencingNGSNext generation sequencingTGSThird generation sequencingTHBGDTibetan hulless barley Genome Database

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September 22, 2019  |  

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

Gene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.We performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.We identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.Discovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.

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September 22, 2019  |  

Resolving the complexity of human skin metagenomes using single-molecule sequencing.

Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation.The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community. Copyright © 2016 Tsai et al.

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September 22, 2019  |  

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed.The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species.The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

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September 22, 2019  |  

Extreme haplotype variation in the desiccation-tolerant clubmoss Selaginella lepidophylla.

Plant genome size varies by four orders of magnitude, and most of this variation stems from dynamic changes in repetitive DNA content. Here we report the small 109?Mb genome of Selaginella lepidophylla, a clubmoss with extreme desiccation tolerance. Single-molecule sequencing enables accurate haplotype assembly of a single heterozygous S. lepidophylla plant, revealing extensive structural variation. We observe numerous haplotype-specific deletions consisting of largely repetitive and heavily methylated sequences, with enrichment in young Gypsy LTR retrotransposons. Such elements are active but rapidly deleted, suggesting “bloat and purge” to maintain a small genome size. Unlike all other land plant lineages, Selaginella has no evidence of a whole-genome duplication event in its evolutionary history, but instead shows unique tandem gene duplication patterns reflecting adaptation to extreme drying. Gene expression changes during desiccation in S. lepidophylla mirror patterns observed across angiosperm resurrection plants.

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September 22, 2019  |  

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

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September 22, 2019  |  

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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