scholarly journals Long-read sequencing and de novo genome assembly of marine medaka (Oryzias melastigma)

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Pingping Liang ◽  
Hafiz Sohaib Ahmed Saqib ◽  
Xiaomin Ni ◽  
Yingjia Shen
Keyword(s):  
Dna Repair ◽  
Positive Selection ◽  
Single Molecule ◽  
Genome Assembly ◽  
De Novo ◽  
Gene Families ◽  
Comparative Genomic ◽  
De Novo Genome Assembly ◽  
Marine Medaka ◽  
Oryzias Melastigma

Abstract Background Marine medaka (Oryzias melastigma) is considered as an important ecotoxicological indicator to study the biochemical, physiological and molecular responses of marine organisms towards increasing amount of pollutants in marine and estuarine waters. Results In this study, we reported a high-quality and accurate de novo genome assembly of marine medaka through the integration of single-molecule sequencing, Illumina paired-end sequencing, and 10X Genomics linked-reads. The 844.17 Mb assembly is estimated to cover more than 98% of the genome and is more continuous with fewer gaps and errors than the previous genome assembly. Comparison of O. melastigma with closely related species showed significant expansion of gene families associated with DNA repair and ATP-binding cassette (ABC) transporter pathways. We identified 274 genes that appear to be under significant positive selection and are involved in DNA repair, cellular transportation processes, conservation and stability of the genome. The positive selection of genes and the considerable expansion in gene numbers, especially related to stimulus responses provide strong supports for adaptations of O. melastigma under varying environmental stresses. Conclusions The highly contiguous marine medaka genome and comparative genomic analyses will increase our understanding of the underlying mechanisms related to its extraordinary adaptation capability, leading towards acceleration in the ongoing and future investigations in marine ecotoxicology.

scholarly journals SMRT sequencing of the Oryza rufipogon genome reveals the genomic basis of rice adaptation

2020 ◽  
Author(s):  
Wei Li ◽  
Kui Li ◽  
Ying Huang ◽  
Cong Shi ◽  
Wu-Shu Hu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Wild Rice ◽  
De Novo ◽  
Rice Genome ◽  
Gene Families ◽  
Oryza Rufipogon ◽  
Genomic Diversity ◽  
Comparative Genomic ◽  
Cultivated Rice ◽  
Reproductive Process

AbstractAsian cultivated rice is believed to have been domesticated from an immediate ancestral progenitor, Oryza rufipogon, which provides promising sources of novel alleles for world rice improvement. Here we first present a high-quality de novo assembly of the typical O. rufipogon genome through the integration of single-molecule sequencing (SMRT), 10× and Hi-C technologies. This chromosome-based reference genome allows a multi-species comparative analysis of the annual selfing O. sativa and its two wild progenitors, the annual selfing O. nivara and perennial outcrossing O. rufipogon, identifying massive numbers of dispensable genes that are functionally enriched in reproductive process. Comparative genomic analyses identified millions of genomic variants, of which large-effect mutations (e.g., SVs, CNV and PAVs) may affect the variation of agronomically significant traits. We demonstrate how lineage-specific expansion of rice gene families may have contributed to the formation of reproduction isolation (e.g., the recognition of pollen and male sterility), thus brightening the role in driving mating system evolution during the evolutionary process of recent speciation. We document thousands of positively selected genes that are mainly involved in flower development, ripening, pollination, reproduction and response to biotic- and abiotic stresses. We show that selection pressures may serve as crucial forces to govern substantial genomic alterations among the three rice species that form the genetic basis of rapid evolution of mating and reproductive systems under diverse habitats. This first chromosome-based wild rice genome in the genus Oryza will become powerful to accelerate the exploration of untapped genomic diversity from wild rice for the enhancement of elite rice cultivars.

scholarly journals Hybrid de novo Genome Assembly of Erwinia sp. E602 and Bioinformatic Analysis Characterized a New Plasmid-Borne lac Operon Under Positive Selection

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu Xia ◽  
Zhi-Yuan Wei ◽  
Rui He ◽  
Jia-Huan Li ◽  
Zhi-Xin Wang ◽  
...  
Keyword(s):  
Positive Selection ◽  
Genome Assembly ◽  
De Novo ◽  
Bioinformatic Analysis ◽  
Lac Operon ◽  
Pacbio Sequencing ◽  
Metabolic Pathway Analysis ◽  
De Novo Genome Assembly ◽  
Sequencing Technologies ◽  
Lactose Metabolism

Our previous study identified a new β-galactosidase in Erwinia sp. E602. To further understand the lactose metabolism in this strain, de novo genome assembly was conducted by using a strategy combining Illumina and PacBio sequencing technology. The whole genome of Erwinia sp. E602 includes a 4.8 Mb chromosome and a 326 kb large plasmid. A total of 4,739 genes, including 4,543 protein-coding genes, 25 rRNAs, 82 tRNAs and 7 other ncRNAs genes were annotated. The plasmid was the largest one characterized in genus Erwinia by far, and it contained a number of genes and pathways responsible for lactose metabolism and regulation. Moreover, a new plasmid-borne lac operon that lacked a typical β-galactoside transacetylase (lacA) gene was identified in the strain. Phylogenetic analysis showed that the genes lacY and lacZ in the operon were under positive selection, indicating the adaptation of lactose metabolism to the environment in Erwinia sp. E602. Our current study demonstrated that the hybrid de novo genome assembly using Illumina and PacBio sequencing technologies, as well as the metabolic pathway analysis, provided a useful strategy for better understanding of the evolution of undiscovered microbial species or strains.

scholarly journals A High-Quality De Novo Genome Assembly from a Single Mosquito using PacBio Sequencing

2018 ◽  
Author(s):  
Sarah B. Kingan ◽  
Haynes Heaton ◽  
Juliana Cudini ◽  
Christine C. Lambert ◽  
Primo Baybayan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Population Genomics ◽  
De Novo ◽  
Anopheles Coluzzii ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Core Technology ◽  
Conserved Genes ◽  
Diploid Individual

AbstractA high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (∼5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 hour movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes are present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

scholarly journals Fully Phased Sequence of a Diploid Human Genome Determined de Novo from the DNA of a Single Individual

2020 ◽  
Vol 10 (9) ◽  
pp. 2911-2925
Author(s):  
llya Soifer ◽  
Nicole L Fong ◽  
Nelda Yi ◽  
Andrea T Ireland ◽  
Irene Lam ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Phenotypic Trait ◽  
Single Individual ◽  
Phase Information ◽  
Metaphase Chromosomes ◽  
De Novo Genome Assembly ◽  
Diploid Genome

Abstract In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

scholarly journals A High-Quality De novo Genome Assembly from a Single Mosquito Using PacBio Sequencing

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 62 ◽  
Author(s):  
Sarah Kingan ◽  
Haynes Heaton ◽  
Juliana Cudini ◽  
Christine Lambert ◽  
Primo Baybayan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Population Genomics ◽  
De Novo ◽  
Anopheles Coluzzii ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Core Technology ◽  
Conserved Genes ◽  
Diploid Individual

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

scholarly journals High-throughput long paired-end sequencing of a Fosmid library by PacBio

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Zhaozhao Dai ◽  
Tong Li ◽  
Jiadong Li ◽  
Zhifei Han ◽  
Yonglong Pan ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Single Molecule ◽  
Genome Assembly ◽  
De Novo ◽  
Average Length ◽  
New Method ◽  
Segmental Duplications ◽  
Structural Rearrangements ◽  
De Novo Genome Assembly ◽  
Paired End Sequencing

Abstract Background Large insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries. Results A Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~ 8 kb) to be fragmented into 2–3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5–10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations. Conclusions We developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

scholarly journals De novo genome assembly of the endangered Acer yangbiense, a plant species with extremely small populations endemic to Yunnan Province, China

GigaScience ◽  
2019 ◽  
Vol 8 (7) ◽  
Author(s):  
Jing Yang ◽  
Hafiz Muhammad Wariss ◽  
Lidan Tao ◽  
Rengang Zhang ◽  
Quanzheng Yun ◽  
...  
Keyword(s):  
Plant Species ◽  
Single Molecule ◽  
Dna Sequences ◽  
Genome Assembly ◽  
Yunnan Province ◽  
De Novo ◽  
Small Populations ◽  
High Quality ◽  
Illumina Hiseq ◽  
De Novo Genome Assembly

Abstract Background Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”. Findings We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ∼666 Mb, with 13 chromosomes covering ∼97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization. Conclusion Here, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales.

scholarly journals A high-quality de novo genome assembly of one swamp eel (Monopterus albus) strain with PacBio and Hi-C sequencing data

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hai-Feng Tian ◽  
Qiao-Mu Hu ◽  
Zhong Li
Keyword(s):  
Single Molecule ◽  
Selective Breeding ◽  
Reference Genome ◽  
De Novo ◽  
Gene Families ◽  
Sequencing Data ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Monopterus Albus ◽  
Swamp Eel

Abstract The swamp eel (Monopterus albus) is one economically important fish in China and South-Eastern Asia and a good model species to study sex inversion. There are different genetic lineages and multiple local strains of swamp eel in China, and one local strain of M. albus with deep yellow and big spots has been selected for consecutive selective breeding due to superiority in growth rate and fecundity. A high-quality reference genome of the swamp eel would be a very useful resource for future selective breeding program. In the present study, we applied PacBio single-molecule sequencing technique (SMRT) and the high-throughput chromosome conformation capture (Hi-C) technologies to assemble the M. albus genome. A 799 Mb genome was obtained with the contig N50 length of 2.4 Mb and scaffold N50 length of 67.24 Mb, indicating 110-fold and ∼31.87-fold improvement compared to the earlier released assembly (∼22.24 Kb and 2.11 Mb, respectively). Aided with Hi-C data, a total of 750 contigs were reliably assembled into 12 chromosomes. Using 22,373 protein-coding genes annotated here, the phylogenetic relationships of the swamp eel with other teleosts showed that swamp eel separated from the common ancestor of Zig-zag eel ∼49.9 million years ago, and 769 gene families were found expanded, which are mainly enriched in the immune system, sensory system, and transport and catabolism. This highly accurate, chromosome-level reference genome of M. albus obtained in this work will be used for the development of genome-scale selective breeding.

scholarly journals Human Genome Assembly in 100 Minutes

2019 ◽  
Author(s):  
Chen-Shan Chin ◽  
Asif Khalak
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Genome Assembly ◽  
De Novo ◽  
Critical Factor ◽  
Read Length ◽  
De Novo Genome Assembly ◽  
Small Indels ◽  
Sequencing Technologies ◽  
Long Read

AbstractDe novo genome assembly provides comprehensive, unbiased genomic information and makes it possible to gain insight into new DNA sequences not present in reference genomes. Many de novo human genomes have been published in the last few years, leveraging a combination of inexpensive short-read and single-molecule long-read technologies. As long-read DNA sequencers become more prevalent, the computational burden of generating assemblies persists as a critical factor. The most common approach to long-read assembly, using an overlap-layout-consensus (OLC) paradigm, requires all-to-all read comparisons, which quadratically scales in computational complexity with the number of reads. We assert that recently achievements in sequencing technology (i.e. with accuracy ~99% and read length ~10-15k) enables a fundamentally better strategy for OLC that is effectively linear rather than quadratic. Our genome assembly implementation, Peregrine uses sparse hierarchical minimizers (SHIMMER) to index reads thereby avoiding the need for an all-to-all read comparison step. Peregrine can assemble 30x human PacBio CCS read datasets in less than 30 CPU hours and around 100 wall-clock minutes to a high contiguity assembly (N50 > 20Mb). The continued advance of sequencing technologies coupled with the Peregrine assembler enables routine generation of human de novo assemblies. This will allow for population scale measurements of more comprehensive genomic variations -- beyond SNPs and small indels -- as well as novel applications requiring rapid access to de novo assemblies.

scholarly journals SMARTdenovo: A de novo Assembler Using Long Noisy Reads

2020 ◽  
Author(s):  
Hailin Liu ◽  
Shigang Wu ◽  
Alun Li ◽  
Jue Ruan
Keyword(s):  
Error Correction ◽  
Single Molecule ◽  
Genome Assembly ◽  
De Novo ◽  
Structural Variants ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Single Molecule Sequencing ◽  
Long Read ◽  
Reference Quality

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. It also has been widely used to study structural variants, phase haplotypes and more. Here, we introduce the assembler— SMARTdenovo, which is an SMS assembler that follows the overlap-layout-consensus (OLC) paradigm. SMARTdenovo (RRID: SCR_017622) was designed to be a fast assembler that did not require highly accurate raw reads for error correction, unlike other, contemporaneous SMS assemblers. It has performed well for evaluating congeneric assemblers and has been successful for a variety of assembly projects. It is compatible with Canu for assembling high-quality genomes, and several of the assembly strategies in this program have been incorporated into subsequent popular assemblers. The assembler has been in use since 2015, and here we provide information on the development of SMARTdenovo and how to implement its algorithms into current projects.

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