scholarly journals Nanopore sequencing significantly improves genome assembly of the eukaryotic protozoan parasite Trypanosoma cruzi

2018 ◽  
Author(s):  
Florencia Diaz-Viraque ◽  
Sebastian Pita ◽  
Gonzalo Greif ◽  
Rita de Cassia Moreira de Souza ◽  
Gregorio Iraola ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Genome Assembly ◽  
De Novo ◽  
Repetitive Sequences ◽  
Protozoan Parasite ◽  
Single Copy ◽  
Nanopore Sequencing ◽  
Short Reads ◽  
Comparative Analyses ◽  
Long Reads

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a two-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ~16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource since it now represents the best-quality assembly available for TcII, a highly prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of T. cruzi and facilitates more comprehensive evolutionary inferences. Our work represents the first report of Nanopore technology used to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.

scholarly journals Nanopore Sequencing Significantly Improves Genome Assembly of the Protozoan Parasite Trypanosoma cruzi

2019 ◽  
Vol 11 (7) ◽  
pp. 1952-1957 ◽  
Author(s):  
Florencia Díaz-Viraqué ◽  
Sebastián Pita ◽  
Gonzalo Greif ◽  
Rita de Cássia Moreira de Souza ◽  
Gregorio Iraola ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Genome Assembly ◽  
De Novo ◽  
Repetitive Sequences ◽  
Protozoan Parasite ◽  
Single Copy ◽  
Nanopore Sequencing ◽  
Short Reads ◽  
Comparative Analyses ◽  
Clade C

Abstract Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.

scholarly journals De novo Genome Assembly of the indica Rice Variety IR64 Using Linked-Read Sequencing and Nanopore Sequencing

2020 ◽  
Vol 10 (5) ◽  
pp. 1495-1501 ◽  
Author(s):  
Tsuyoshi Tanaka ◽  
Ryo Nishijima ◽  
Shota Teramoto ◽  
Yuka Kitomi ◽  
Takeshi Hayashi ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Genome Assembly ◽  
De Novo ◽  
Rice Variety ◽  
Rice Genome ◽  
High Yield ◽  
Nanopore Sequencing ◽  
Long Reads ◽  
A Genome ◽  
Modern Varieties

IR64 is a rice variety with high-yield that has been widely cultivated around the world. IR64 has been replaced by modern varieties in most growing areas. Given that modern varieties are mostly progenies or relatives of IR64, genetic analysis of IR64 is valuable for rice functional genomics. However, chromosome-level genome sequences of IR64 have not been available previously. Here, we sequenced the IR64 genome using synthetic long reads obtained by linked-read sequencing and ultra-long reads obtained by nanopore sequencing. We integrated these data and generated the de novo assembly of the IR64 genome of 367 Mb, equivalent to 99% of the estimated size. Continuity of the IR64 genome assembly was improved compared with that of a publicly available IR64 genome assembly generated by short reads only. We annotated 41,458 protein-coding genes, including 657 IR64-specific genes, that are missing in other high-quality rice genome assemblies IRGSP-1.0 of japonica cultivar Nipponbare or R498 of indica cultivar Shuhui498. The IR64 genome assembly will serve as a genome resource for rice functional genomics as well as genomics-driven and/or molecular breeding.

scholarly journals The First Draft Genome Assembly of Snow Sheep (Ovis nivicola)

2020 ◽  
Vol 12 (8) ◽  
pp. 1330-1336 ◽  
Author(s):  
Maulik Upadhyay ◽  
Andreas Hauser ◽  
Elisabeth Kunz ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
...  
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
Gene Prediction ◽  
Repetitive Sequences ◽  
Draft Genome ◽  
Single Copy ◽  
Climatic Conditions ◽  
Draft Genome Assembly ◽  
Sheep Genome ◽  
Long Reads

Abstract The snow sheep, Ovis nivicola, which is endemic to the mountain ranges of northeastern Siberia, are well adapted to the harsh cold climatic conditions of their habitat. In this study, using long reads of Nanopore sequencing technology, whole-genome sequencing, assembly, and gene annotation of a snow sheep were carried out. Additionally, RNA-seq reads from several tissues were also generated to supplement the gene prediction in snow sheep genome. The assembled genome was ∼2.62 Gb in length and was represented by 7,157 scaffolds with N50 of about 2 Mb. The repetitive sequences comprised of 41% of the total genome. BUSCO analysis revealed that the snow sheep assembly contained full-length or partial fragments of 97% of mammalian universal single-copy orthologs (n = 4,104), illustrating the completeness of the assembly. In addition, a total of 20,045 protein-coding sequences were identified using comprehensive gene prediction pipeline. Of which 19,240 (∼96%) sequences were annotated using protein databases. Moreover, homology-based searches and de novo identification detected 1,484 tRNAs; 243 rRNAs; 1,931 snRNAs; and 782 miRNAs in the snow sheep genome. To conclude, we generated the first de novo genome of the snow sheep using long reads; these data are expected to contribute significantly to our understanding related to evolution and adaptation within the Ovis genus.

scholarly journals Draft genome of a porcupinefish, Diodon Holocanthus

2019 ◽  
Author(s):  
Mengyang Xu ◽  
Xiaoshan Su ◽  
Mengqi Zhang ◽  
Ming Li ◽  
Xiaoyun Huang ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Repetitive Sequences ◽  
Draft Genome ◽  
Single Copy ◽  
Single Individual ◽  
Protein Coding ◽  
Long Read ◽  
Phylogeny And Evolution ◽  
Downstream Analysis

AbstractThe long-spine porcupinefish, Diodon holocanthus (Diodontidae, Tetraodontiformes, Actinopterygii), also known as the freckled porcupinefish, attracts great interest of ecology and economy. Its distinct characteristics including inflation reaction, spiny skin and tetradotoxin, however, have not been fully studied without a complete genome assembly.In this study, the whole genome of a single individual was sequenced using single tube-Long Fragment Read co-barcode reads, generating 154.3 Gb of paired-end data (219.8× depth). The gap was further filled using small amount of Oxford Nanopore MinION long read dataset (11.4Gb, 15.9× depth). Taking full use of long, medium, short-range of genome assembly information, the final assembled sequences with a total length of 650.02 Mb obtained contig and scaffold N50 sizes of 2.15 Mb and 8.13 Mb, respectively, despite of high repetitive content. Benchmarking Universal Single-Copy Orthologs captured 95.7% (2,474) of core genes to assess the completeness. In addition, 206.5 Mb (32.10%) of repetitive sequences were identified, and 20,840 protein-coding genes were annotated, among which 18,281 (87.72%) proteins were assigned with possible functions.This is the first demonstration of de novo genome of the porcupinefish, which will benefit downstream analysis of ontogeny, phylogeny, and evolution, and improve the exploration of its unique defensive mechanism.

scholarly journals A de novo Full-Length mRNA Transcriptome Generated From Hybrid-Corrected PacBio Long-Reads Improves the Transcript Annotation and Identifies Thousands of Novel Splice Variants in Atlantic Salmon

2021 ◽  
Vol 12 ◽  
Author(s):  
Sigmund Ramberg ◽  
Bjørn Høyheim ◽  
Tone-Kari Knutsdatter Østbye ◽  
Rune Andreassen
Keyword(s):  
Atlantic Salmon ◽  
Salmo Salar ◽  
Genome Assembly ◽  
De Novo ◽  
Splice Variants ◽  
Full Length ◽  
Short Reads ◽  
Long Reads ◽  
Long Read ◽  
Mrna Transcriptome

Atlantic salmon (Salmo salar) is a major species produced in world aquaculture and an important vertebrate model organism for studying the process of rediploidization following whole genome duplication events (Ss4R, 80 mya). The current Salmo salar transcriptome is largely generated from genome sequence based in silico predictions supported by ESTs and short-read sequencing data. However, recent progress in long-read sequencing technologies now allows for full-length transcript sequencing from single RNA-molecules. This study provides a de novo full-length mRNA transcriptome from liver, head-kidney and gill materials. A pipeline was developed based on Iso-seq sequencing of long-reads on the PacBio platform (HQ reads) followed by error-correction of the HQ reads by short-reads from the Illumina platform. The pipeline successfully processed more than 1.5 million long-reads and more than 900 million short-reads into error-corrected HQ reads. A surprisingly high percentage (32%) represented expressed interspersed repeats, while the remaining were processed into 71 461 full-length mRNAs from 23 071 loci. Each transcript was supported by several single-molecule long-read sequences and at least three short-reads, assuring a high sequence accuracy. On average, each gene was represented by three isoforms. Comparisons to the current Atlantic salmon transcripts in the RefSeq database showed that the long-read transcriptome validated 25% of all known transcripts, while the remaining full-length transcripts were novel isoforms, but few were transcripts from novel genes. A comparison to the current genome assembly indicates that the long-read transcriptome may aid in improving transcript annotation as well as provide long-read linkage information useful for improving the genome assembly. More than 80% of transcripts were assigned GO terms and thousands of transcripts were from genes or splice-variants expressed in an organ-specific manner demonstrating that hybrid error-corrected long-read transcriptomes may be applied to study genes and splice-variants expressed in certain organs or conditions (e.g., challenge materials). In conclusion, this is the single largest contribution of full-length mRNAs in Atlantic salmon. The results will be of great value to salmon genomics research, and the pipeline outlined may be applied to generate additional de novo transcriptomes in Atlantic Salmon or applied for similar projects in other species.

scholarly journals A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout

2021 ◽  
Author(s):  
Guangtu Gao ◽  
Susana Magadan ◽  
Geoffrey C Waldbieser ◽  
Ramey C Youngblood ◽  
Paul A Wheeler ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Chromosome Number ◽  
Genome Assembly ◽  
De Novo Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Structural Variations ◽  
High Coverage ◽  
Haploid Chromosome Number ◽  
Long Reads

Abstract Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.

scholarly journals De Novo Assembly of a High-Quality Reference Genome for the Horned Lark (Eremophila alpestris)

2019 ◽  
Vol 10 (2) ◽  
pp. 475-478 ◽  
Author(s):  
Nicholas A. Mason ◽  
Paulo Pulgarin ◽  
Carlos Daniel Cadena ◽  
Irby J. Lovette
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Single Copy ◽  
Single Copy Gene ◽  
High Quality ◽  
Data Set ◽  
Copy Gene ◽  
Assembly Pipeline ◽  
Horned Lark ◽  
Gene Orthologs

The Horned Lark (Eremophila alpestris) is a small songbird that exhibits remarkable geographic variation in appearance and habitat across an expansive distribution. While E. alpestris has been the focus of many ecological and evolutionary studies, we still lack a highly contiguous genome assembly for the Horned Lark and related taxa (Alaudidae). Here, we present CLO_EAlp_1.0, a highly contiguous assembly for E. alpestris generated from a blood sample of a wild, male bird captured in the Altiplano Cundiboyacense of Colombia. By combining short-insert and mate-pair libraries with the ALLPATHS-LG genome assembly pipeline, we generated a 1.04 Gb assembly comprised of 2713 scaffolds, with a largest scaffold size of 31.81 Mb, a scaffold N50 of 9.42 Mb, and a scaffold L50 of 30. These scaffolds were assembled from 23685 contigs, with a largest contig size of 1.69 Mb, a contig N50 of 193.81 kb, and a contig L50 of 1429. Our assembly pipeline also produced a single mitochondrial DNA contig of 14.00 kb. After polishing the genome, we identified 94.5% of single-copy gene orthologs from an Aves data set and 97.7% of single-copy gene orthologs from a vertebrata data set, which further demonstrates the high quality of our assembly. We anticipate that this genomic resource will be useful to the broader ornithological community and those interested in studying the evolutionary history and ecological interactions of larks, which comprise a widespread, yet understudied lineage of songbirds.

scholarly journals A haplotype-resolved, de novo genome assembly for the wood tiger moth (Arctia plantaginis) through trio binning

GigaScience ◽  
2020 ◽  
Vol 9 (8) ◽  
Author(s):  
Eugenie C Yen ◽  
Shane A McCarthy ◽  
Juan A Galarza ◽  
Tomas N Generalovic ◽  
Sarah Pelan ◽  
...  
Keyword(s):  
Population Structure ◽  
Genome Assembly ◽  
De Novo ◽  
Consensus Sequence ◽  
Parental Origin ◽  
De Novo Genome Assembly ◽  
Geographic Population ◽  
Long Reads ◽  
Tiger Moth ◽  
Innovative Solution

ABSTRACT Background Diploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence. Trio binning offers an innovative solution that exploits heterozygosity for assembly. Short, parental reads are used to assign parental origin to long reads from their F1 offspring before assembly, enabling complete haplotype resolution. Trio binning could therefore provide an effective strategy for assembling highly heterozygous genomes, which are traditionally problematic, such as insect genomes. This includes the wood tiger moth (Arctia plantaginis), which is an evolutionary study system for warning colour polymorphism. Findings We produced a high-quality, haplotype-resolved assembly for Arctia plantaginis through trio binning. We sequenced a same-species family (F1 heterozygosity ∼1.9%) and used parental Illumina reads to bin 99.98% of offspring Pacific Biosciences reads by parental origin, before assembling each haplotype separately and scaffolding with 10X linked reads. Both assemblies are contiguous (mean scaffold N50: 8.2 Mb) and complete (mean BUSCO completeness: 97.3%), with annotations and 31 chromosomes identified through karyotyping. We used the assembly to analyse genome-wide population structure and relationships between 40 wild resequenced individuals from 5 populations across Europe, revealing the Georgian population as the most genetically differentiated with the lowest genetic diversity. Conclusions We present the first invertebrate genome to be assembled via trio binning. This assembly is one of the highest quality genomes available for Lepidoptera, supporting trio binning as a potent strategy for assembling heterozygous genomes. Using our assembly, we provide genomic insights into the geographic population structure of A. plantaginis.

scholarly journals De Novo Sequencing and Hybrid Assembly of the Biofuel Crop Jatropha curcas L.: Identification of Quantitative Trait Loci for Geminivirus Resistance

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 69 ◽  
Author(s):  
Nagesh Kancharla ◽  
Saakshi Jalali ◽  
J. Narasimham ◽  
Vinod Nair ◽  
Vijay Yepuri ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Genome Assembly ◽  
Jatropha Curcas ◽  
Quantitative Trait ◽  
De Novo ◽  
Mapping Population ◽  
Single Copy ◽  
Sequencing Data ◽  
De Novo Genome Assembly ◽  
Sequencing Technologies

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines.

scholarly journals Whole-Genome Sequencing of the Giant Devil Catfish, Bagarius yarrelli

2019 ◽  
Vol 11 (8) ◽  
pp. 2071-2077 ◽  
Author(s):  
Wansheng Jiang ◽  
Yunyun Lv ◽  
Le Cheng ◽  
Kunfeng Yang ◽  
Chao Bian ◽  
...  
Keyword(s):  
Body Size ◽  
Ictalurus Punctatus ◽  
Genome Assembly ◽  
De Novo ◽  
Large Body ◽  
Single Copy ◽  
The Body ◽  
Flesh Color ◽  
Fish Muscles ◽  
Freshwater Aquaculture

AbstractAs one economically important fish in the southeastern Himalayas, the giant devil catfish (Bagarius yarrelli) has been known for its extraordinarily large body size. It can grow up to 2 m, whereas the non-Bagarius sisorids only reach 10–30 cm. Another outstanding characteristic of Bagarius species is the salmonids-like reddish flesh color. Both body size and flesh color are interesting questions in science and also valuable features in aquaculture that worth of deep investigations. Bagarius species therefore are ideal materials for studying body size evolution and color depositions in fish muscles, and also potential organisms for extensive utilization in Asian freshwater aquaculture. In a combination of Illumina and PacBio sequencing technologies, we de novo assembled a 571-Mb genome for the giant devil catfish from a total of 153.4-Gb clean reads. The scaffold and contig N50 values are 3.1 and 1.6 Mb, respectively. This genome assembly was evaluated with 93.4% of Benchmarking Universal Single-Copy Orthologs completeness, 98% of transcripts coverage, and highly homologous with a chromosome-level-based genome of channel catfish (Ictalurus punctatus). We detected that 35.26% of the genome assembly is composed of repetitive elements. Employing homology, de novo, and transcriptome-based annotations, we annotated a total of 19,027 protein-coding genes for further use. In summary, we generated the first high-quality genome assembly of the giant devil catfish, which provides an important genomic resource for its future studies such as the body size and flesh color issues, and also for facilitating the conservation and utilization of this valuable catfish.

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