Auto Tag: long read error correction

April 21, 2020  |  

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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April 21, 2020  |  

A comprehensive evaluation of long read error correction methods

Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods, and provide a comprehensive evaluation of the corresponding long read error correction tools. Leveraging recent real sequencing data, we establish benchmark data sets and set up evaluation criteria for a comparative assessment which includes quality of error correction as well as run-time and memory usage. We study how trimming and long read sequencing depth affect error correction in terms of length distribution and genome coverage post-correction, and the impact of error correction performance on an important application of long reads, genome assembly. We provide guidelines for practitioners for choosing among the available error correction tools and identify directions for future research.

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April 21, 2020  |  

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Pseudomolecule-level assembly of the Chinese oil tree yellowhorn (Xanthoceras sorbifolium) genome.

Yellowhorn (Xanthoceras sorbifolium) is a species of the Sapindaceae family native to China and is an oil tree that can withstand cold and drought conditions. A pseudomolecule-level genome assembly for this species will not only contribute to understanding the evolution of its genes and chromosomes but also bring yellowhorn breeding into the genomic era.Here, we generated 15 pseudomolecules of yellowhorn chromosomes, on which 97.04% of scaffolds were anchored, using the combined Illumina HiSeq, Pacific Biosciences Sequel, and Hi-C technologies. The length of the final yellowhorn genome assembly was 504.2 Mb with a contig N50 size of 1.04 Mb and a scaffold N50 size of 32.17 Mb. Genome annotation revealed that 68.67% of the yellowhorn genome was composed of repetitive elements. Gene modelling predicted 24,672 protein-coding genes. By comparing orthologous genes, the divergence time of yellowhorn and its close sister species longan (Dimocarpus longan) was estimated at ~33.07 million years ago. Gene cluster and chromosome synteny analysis demonstrated that the yellowhorn genome shared a conserved genome structure with its ancestor in some chromosomes.This genome assembly represents a high-quality reference genome for yellowhorn. Integrated genome annotations provide a valuable dataset for genetic and molecular research in this species. We did not detect whole-genome duplication in the genome. The yellowhorn genome carries syntenic blocks from ancient chromosomes. These data sources will enable this genome to serve as an initial platform for breeding better yellowhorn cultivars. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

The Genome of Armadillidium vulgare (Crustacea, Isopoda) Provides Insights into Sex Chromosome Evolution in the Context of Cytoplasmic Sex Determination.

The terrestrial isopod Armadillidium vulgare is an original model to study the evolution of sex determination and symbiosis in animals. Its sex can be determined by ZW sex chromosomes, or by feminizing Wolbachia bacterial endosymbionts. Here, we report the sequence and analysis of the ZW female genome of A. vulgare. A distinguishing feature of the 1.72 gigabase assembly is the abundance of repeats (68% of the genome). We show that the Z and W sex chromosomes are essentially undifferentiated at the molecular level and the W-specific region is extremely small (at most several hundreds of kilobases). Our results suggest that recombination suppression has not spread very far from the sex-determining locus, if at all. This is consistent with A. vulgare possessing evolutionarily young sex chromosomes. We characterized multiple Wolbachia nuclear inserts in the A. vulgare genome, none of which is associated with the W-specific region. We also identified several candidate genes that may be involved in the sex determination or sexual differentiation pathways. The A. vulgare genome serves as a resource for studying the biology and evolution of crustaceans, one of the most speciose and emblematic metazoan groups. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Genome sequence and genetic transformation of a widely distributed and cultivated poplar.

Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein-coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Efficiency of PacBio long read correction by 2nd generation Illumina sequencing.

Long sequencing reads offer unprecedented opportunities in analysis and reconstruction of complex genomic regions. However, the gain in sequence length is often traded for quality. Therefore, recently several approaches have been proposed (e.g. higher sequencing coverage, hybrid assembly or sequence correction) to enhance the quality of long sequencing reads. A simple and cost-effective approach includes use of the high quality 2nd generation sequencing data to improve the quality of long reads. We designed a dedicated testing procedure and selected universal programs for long read correction, which provide as the output sequences that can be used in further genomic and transcriptomic studies. Our results show that HALC is the best choice for correction of long PacBio reads, when both, read size and quality, are the main focus of the analysis. However, the tested tools show some unexpected behaviors, including read trimming and fragmentation.Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Full-length transcriptome analysis of Litopenaeus vannamei reveals transcript variants involved in the innate immune system.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Computational aspects underlying genome to phenome analysis in plants.

Recent advances in genomics technologies have greatly accelerated the progress in both fundamental plant science and applied breeding research. Concurrently, high-throughput plant phenotyping is becoming widely adopted in the plant community, promising to alleviate the phenotypic bottleneck. While these technological breakthroughs are significantly accelerating quantitative trait locus (QTL) and causal gene identification, challenges to enable even more sophisticated analyses remain. In particular, care needs to be taken to standardize, describe and conduct experiments robustly while relying on plant physiology expertise. In this article, we review the state of the art regarding genome assembly and the future potential of pangenomics in plant research. We also describe the necessity of standardizing and describing phenotypic studies using the Minimum Information About a Plant Phenotyping Experiment (MIAPPE) standard to enable the reuse and integration of phenotypic data. In addition, we show how deep phenotypic data might yield novel trait-trait correlations and review how to link phenotypic data to genomic data. Finally, we provide perspectives on the golden future of machine learning and their potential in linking phenotypes to genomic features. © 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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April 21, 2020  |  

Diploid Genome Assembly of the Wine Grape Carménère.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.Copyright © 2019 Minio et al.

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April 21, 2020  |  

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

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April 21, 2020  |  

Whole genome sequence and de novo assembly revealed genomic architecture of Indian Mithun (Bos frontalis).

Mithun (Bos frontalis), also called gayal, is an endangered bovine species, under the tribe bovini with 2n?=?58 XX chromosome complements and reared under the tropical rain forests region of India, China, Myanmar, Bhutan and Bangladesh. However, the origin of this species is still disputed and information on its genomic architecture is scanty so far. We trust that availability of its whole genome sequence data and assembly will greatly solve this problem and help to generate many information including phylogenetic status of mithun. Recently, the first genome assembly of gayal, mithun of Chinese origin, was published. However, an improved reference genome assembly would still benefit in understanding genetic variation in mithun populations reared under diverse geographical locations and for building a superior consensus assembly. We, therefore, performed deep sequencing of the genome of an adult female mithun from India, assembled and annotated its genome and performed extensive bioinformatic analyses to produce a superior de novo genome assembly of mithun.We generated ˜300 Gigabyte (Gb) raw reads from whole-genome deep sequencing platforms and assembled the sequence data using a hybrid assembly strategy to create a high quality de novo assembly of mithun with 96% recovered as per BUSCO analysis. The final genome assembly has a total length of 3.0 Gb, contains 5,015 scaffolds with an N50 value of 1?Mb. Repeat sequences constitute around 43.66% of the assembly. The genomic alignments between mithun to cattle showed that their genomes, as expected, are highly conserved. Gene annotation identified 28,044 protein-coding genes presented in mithun genome. The gene orthologous groups of mithun showed a high degree of similarity in comparison with other species, while fewer mithun specific coding sequences were found compared to those in cattle.Here we presented the first de novo draft genome assembly of Indian mithun having better coverage, less fragmented, better annotated, and constitutes a reasonably complete assembly compared to the previously published gayal genome. This comprehensive assembly unravelled the genomic architecture of mithun to a great extent and will provide a reference genome assembly to research community to elucidate the evolutionary history of mithun across its distinct geographical locations.

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April 21, 2020  |  

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

Gossypium australe F. Mueller (2n?=?2x?=?26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

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April 21, 2020  |  

De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission.

The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora.Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides.By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.

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April 21, 2020  |  

A comparative evaluation of hybrid error correction methods for error-prone long reads.

Third-generation sequencing technologies have advanced the progress of the biological research by generating reads that are substantially longer than second-generation sequencing technologies. However, their notorious high error rate impedes straightforward data analysis and limits their application. A handful of error correction methods for these error-prone long reads have been developed to date. The output data quality is very important for downstream analysis, whereas computing resources could limit the utility of some computing-intense tools. There is a lack of standardized assessments for these long-read error-correction methods.Here, we present a comparative performance assessment of ten state-of-the-art error-correction methods for long reads. We established a common set of benchmarks for performance assessment, including sensitivity, accuracy, output rate, alignment rate, output read length, run time, and memory usage, as well as the effects of error correction on two downstream applications of long reads: de novo assembly and resolving haplotype sequences.Taking into account all of these metrics, we provide a suggestive guideline for method choice based on available data size, computing resources, and individual research goals.

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