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 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 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 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.

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 A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

GigaScience ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Sarah B Kingan ◽  
Julie Urban ◽  
Christine C Lambert ◽  
Primo Baybayan ◽  
Anna K Childers ◽  
...  
Keyword(s):  
Invasive Species ◽  
Genome Assembly ◽  
De Novo ◽  
Fragment Size ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Lycorma Delicatula ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome

ABSTRACT Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ∼20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ∼36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

scholarly journals A high-quality de novo genome assembly from a single parasitoid wasp

2020 ◽  
Author(s):  
Xinhai Ye ◽  
Yi Yang ◽  
Zhaoyang Tian ◽  
Le Xu ◽  
Kaili Yu ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Parasitoid Wasp ◽  
Genome Comparison ◽  
Single Individual ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Low Input ◽  
A Genome ◽  
High Level

AbstractSequencing and assembling a genome with a single individual have several advantages, such as lower heterozygosity and easier sample preparation. However, the amount of genomic DNA of some small sized organisms might not meet the standard DNA input requirement for current sequencing pipelines. Although few studies sequenced a single small insect with about 100 ng DNA as input, it may still be challenging for many small organisms to obtain such amount of DNA from a single individual. Here, we use 20 ng DNA as input, and present a high-quality genome assembly for a single haploid male parasitoid wasp (Habrobracon hebetor) using Nanopore and Illumina. Because of the low input DNA, a whole genome amplification (WGA) method is used before sequencing. The assembled genome size is 131.6 Mb with a contig N50 of 1.63 Mb. A total of 99% Benchmarking Universal Single-Copy Orthologs are detected, suggesting the high level of completeness of the genome assembly. Genome comparison between H. hebetor and its relative Bracon brevicornis shows a high-level genome synteny, indicating the genome of H. hebetor is highly accurate and contiguous. Our study provides an example for de novo assembling a genome from ultra-low input DNA, and will be used for sequencing projects of small sized species and rare samples, haploid genomics as well as population genetics of small sized species.

scholarly journals A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

2018 ◽  
Author(s):  
Jolene T. Sutton ◽  
Martin Helmkampf ◽  
Cynthia C. Steiner ◽  
M. Renee Bellinger ◽  
Jonas Korlach ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Captive Breeding ◽  
De Novo ◽  
Bird Species ◽  
Population Level ◽  
Model Systems ◽  
Population Declines ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Long Read

AbstractGenome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, and to apply these results to conservation management. Here we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the Alala. As the only remaining native crow species in Hawaii, the Alala survived solely in a captive breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies, and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important for conservation applications, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

scholarly journals Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima)

GigaScience ◽  
2019 ◽  
Vol 8 (9) ◽  
Author(s):  
Yu Xing ◽  
Yang Liu ◽  
Qing Zhang ◽  
Xinghua Nie ◽  
Yamin Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Genetic Improvement ◽  
De Novo ◽  
Draft Genome ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
High Quality ◽  
Chinese Chestnut ◽  
Castanea Mollissima

AbstractBackgroundThe Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima.FindingsWe produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ∼785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ∼13.62 million years ago.ConclusionsThe high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality.

scholarly journals High-Quality de novo Genome Assembly of Huajingxian 74, a Receptor Parent of Single Segment Substitution Lines

Rice Science ◽  
2021 ◽  
Vol 28 (2) ◽  
pp. 109-113
Author(s):  
Li Fangping ◽  
Gao Yanhao ◽  
Wu Bingqi ◽  
Cai Qingpei ◽  
Zhan Pengling ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Substitution Lines ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Single Segment ◽  
Single Segment Substitution Lines ◽  
Segment Substitution

scholarly journals Efficient long single molecule sequencing for cost effective and accurate sequencing, haplotyping, and de novo assembly

2018 ◽  
Author(s):  
Ou Wang ◽  
Robert Chin ◽  
Xiaofang Cheng ◽  
Michelle Ka Wu ◽  
Qing Mao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
De Novo ◽  
Low Cost ◽  
Variant Calling ◽  
Cost Effective ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Single Tube ◽  
Complex Structural

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

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