scholarly journals Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information

GigaScience ◽  
2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Hui-Su Kim ◽  
Sungwon Jeon ◽  
Changjae Kim ◽  
Yeon Kyung Kim ◽  
Yun Sung Cho ◽  
...  
Keyword(s):  
Human Genome ◽  
Single Molecule ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Low Cost ◽  
Cost Effective ◽  
Sequencing Data ◽  
Smrt Sequencing ◽  
Human Genome Assembly

Abstract Background Long DNA reads produced by single-molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short-read DNA fragments. For de novo assembly, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the favorite options. However, PacBio's SMRT sequencing is expensive for a full human genome assembly and costs more than $40,000 US for 30× coverage as of 2019. ONT PromethION sequencing, on the other hand, is 1/12 the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio's SMRT sequencing in relation to the quality. Findings We performed whole-genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64× coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mb and a total genome length of 2.8 Gb. It was comparable to a KOREF assembly constructed using PacBio at 62× coverage (188 Gb, 2,695 contigs, and N50s of 17.9 Mb). When we applied Hi-C–derived long-range mapping data, an even higher quality assembly for the 64× coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mb. Conclusion The pore-based PromethION approach provided a high-quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and was more cost-effective than PacBio at comparable quality measurements.

scholarly journals Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information

2019 ◽  
Author(s):  
Hui-Su Kim ◽  
Sungwon Jeon ◽  
Changjae Kim ◽  
Yeon Kyung Kim ◽  
Yun Sung Cho ◽  
...  
Keyword(s):  
Human Genome ◽  
Single Molecule ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Low Cost ◽  
Cost Effective ◽  
Sequencing Data ◽  
Smrt Sequencing ◽  
Human Genome Assembly

AbstractBackgroundLong DNA reads produced by single molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short read DNA fragments. For de novo assembly, PacBio and Oxford Nanopore Technologies (ONT) are favorite options. However, PacBio’s SMRT sequencing is expensive for a full human genome assembly and costs over 40,000 USD for 30x coverage as of 2019. ONT PromethION sequencing, on the other hand, is one-twelfth the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio’s SMRT sequencing in relation to the quality.FindingsWe performed whole genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64x coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mbp and a total genome length of 2.8 Gbp. It was comparable to a KOREF assembly constructed using PacBio at 62x coverage (188 Gbp, 2,695 contigs and N50s of 17.9 Mbp). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64x coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mbp.ConclusionThe pore-based PromethION approach provides a good quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and is more cost-effective than PacBio at comparable quality measurements.

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.

AStrap: identification of alternative splicing from transcript sequences without a reference genome

2018 ◽  
Vol 35 (15) ◽  
pp. 2654-2656 ◽  
Author(s):  
Guoli Ji ◽  
Wenbin Ye ◽  
Yaru Su ◽  
Moliang Chen ◽  
Guangzao Huang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Alternative Splicing ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Supplementary Information ◽  
Model Organisms ◽  
Sequencing Data ◽  
Extensive Evaluation ◽  
Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads

2020 ◽  
Author(s):  
David Porubsky ◽  
◽  
Peter Ebert ◽  
Peter A. Audano ◽  
Mitchell R. Vollger ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Assembly ◽  
De Novo ◽  
Error Rates ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
De Novo Genome Assembly ◽  
Parental Data ◽  
Human Genome Assembly ◽  
Long Read

AbstractHuman genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.

scholarly journals GALA: gap-free chromosome-scale assembly with long reads

2020 ◽  
Author(s):  
Mohamed Awad ◽  
Xiangchao Gan
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Genetic Maps ◽  
Sequencing Data ◽  
C Elegans ◽  
Assembly Method ◽  
Long Reads ◽  
Long Read ◽  
Assembly Technology

AbstractHigh-quality genome assembly has wide applications in genetics and medical studies. However, it is still very challenging to achieve gap-free chromosome-scale assemblies using current workflows for long-read platforms. Here we propose GALA (Gap-free long-read assembler), a chromosome-by-chromosome assembly method implemented through a multi-layer computer graph that identifies mis-assemblies within preliminary assemblies or chimeric raw reads and partitions the data into chromosome-scale linkage groups. The subsequent independent assembly of each linkage group generates a gap-free assembly free from the mis-assembly errors which usually hamper existing workflows. This flexible framework also allows us to integrate data from various technologies, such as Hi-C, genetic maps, a reference genome and even motif analyses, to generate gap-free chromosome-scale assemblies. We de novo assembled the C. elegans and A. thaliana genomes using combined Pacbio and Nanopore sequencing data from publicly available datasets. We also demonstrated the new method’s applicability with a gap-free assembly of a human genome with the help a reference genome. In addition, GALA showed promising performance for Pacbio high-fidelity long reads. Thus, our method enables straightforward assembly of genomes with multiple data sources and overcomes barriers that at present restrict the application of de novo genome assembly technology.

scholarly journals W2RAP: a pipeline for high quality, robust assemblies of large complex genomes from short read data

2017 ◽  
Author(s):  
Bernardo J. Clavijo ◽  
Gonzalo Garcia Accinelli ◽  
Jonathan Wright ◽  
Darren Heavens ◽  
Katie Barr ◽  
...  
Keyword(s):  
De Novo ◽  
Low Cost ◽  
Cost Effective ◽  
Data Generation ◽  
Sequencing Data ◽  
High Quality ◽  
Crop Species ◽  
Short Read ◽  
Link Type ◽  
Sequencing Technologies

AbstractProducing high-quality whole-genome shotgun de novo assemblies from plant and animal species with large and complex genomes using low-cost short read sequencing technologies remains a challenge. But when the right sequencing data, with appropriate quality control, is assembled using approaches focused on robustness of the process rather than maximization of a single metric such as the usual contiguity estimators, good quality assemblies with informative value for comparative analyses can be produced. Here we present a complete method described from data generation and qc all the way up to scaffold of complex genomes using Illumina short reads and its application to data from plants and human datasets. We show how to use the w2rap pipeline following a metric-guided approach to produce cost-effective assemblies. The assemblies are highly accurate, provide good coverage of the genome and show good short range contiguity. Our pipeline has already enabled the rapid, cost-effective generation of de novo genome assemblies from large, polyploid crop species with a focus on comparative genomics.Availabilityw2rap is available under MIT license, with some subcomponents under GPL-licenses. A ready-to-run docker with all software pre-requisites and example data is also available.http://github.com/bioinfologics/w2raphttp://github.com/bioinfologics/w2rap-contigger

scholarly journals Chromosome-level reference genome of the European wasp spider Argiope bruennichi: a resource for studies on range expansion and evolutionary adaptation

GigaScience ◽  
2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Monica M Sheffer ◽  
Anica Hoppe ◽  
Henrik Krehenwinkel ◽  
Gabriele Uhl ◽  
Andreas W Kuss ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Range Expansion ◽  
Reference Genome ◽  
De Novo ◽  
Sequencing Data ◽  
High Quality ◽  
Proximity Ligation ◽  
Genomic Resource ◽  
Paired End Sequencing ◽  
Chromosome Level

Abstract Background Argiope bruennichi, the European wasp spider, has been investigated intensively as a focal species for studies on sexual selection, chemical communication, and the dynamics of rapid range expansion at a behavioral and genetic level. However, the lack of a reference genome has limited insights into the genetic basis for these phenomena. Therefore, we assembled a high-quality chromosome-level reference genome of the European wasp spider as a tool for more in-depth future studies. Findings We generated, de novo, a 1.67 Gb genome assembly of A. bruennichi using 21.8× Pacific Biosciences sequencing, polished with 19.8× Illumina paired-end sequencing data, and proximity ligation (Hi-C)-based scaffolding. This resulted in an N50 scaffold size of 124 Mb and an N50 contig size of 288 kb. We found 98.4% of the genome to be contained in 13 scaffolds, fitting the expected number of chromosomes (n = 13). Analyses showed the presence of 91.1% of complete arthropod BUSCOs, indicating a high-quality assembly. Conclusions We present the first chromosome-level genome assembly in the order Araneae. With this genomic resource, we open the door for more precise and informative studies on evolution and adaptation not only in A. bruennichi but also in arachnids overall, shedding light on questions such as the genomic architecture of traits, whole-genome duplication, and the genomic mechanisms behind silk and venom evolution.

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 Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing

2020 ◽  
Author(s):  
Ivan de la Rubia ◽  
Joel A. Indi ◽  
Silvia Carbonell-Sala ◽  
Julien Lagarde ◽  
M Mar Albà ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reference Genome ◽  
Simulated Data ◽  
Cost Effective ◽  
Dna Assembly ◽  
Sequencing Data ◽  
Consensus Sequences ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

AbstractSingle-molecule long-read sequencing with Nanopore provides an unprecedented opportunity to measure transcriptomes from any sample1–3. However, current analysis methods rely on the comparison with a reference genome or transcriptome2,4,5, or the use of multiple sequencing technologies6,7, thereby precluding cost-effective studies in species with no genome assembly available, in individuals underrepresented in the existing reference, and for the discovery of disease-specific transcripts not directly identifiable from a reference genome. Methods for DNA assembly8–10 cannot be directly transferred to transcriptomes since their consensus sequences lack the required interpretability for genes with multiple transcript isoforms. To address these challenges, we have developed RATTLE, the first tool to perform reference-free reconstruction and quantification of transcripts from Nanopore long reads. Using simulated data, isoform spike-ins, and sequencing data from tissues and cell lines, we demonstrate that RATTLE accurately determines transcript sequence and abundance, is comparable to reference-based methods, and shows saturation in the number of predicted transcripts with increasing number of input reads.

scholarly journals Finding Nemo’s Genes: A chromosome-scale reference assembly of the genome of the orange clownfish Amphiprion percula

2018 ◽  
Author(s):  
Robert Lehmann ◽  
Damien J. Lightfoot ◽  
Celia Schunter ◽  
Craig T. Michell ◽  
Hajime Ohyanagi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Reef Fishes ◽  
Population Connectivity ◽  
Adaptation To Climate Change ◽  
Fish Genome ◽  
Amphiprion Percula ◽  
Reference Genome Assembly

AbstractThe iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that anti-predator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here we present a de novo chromosome-scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single-molecule real-time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi-C based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.

Sign in / Sign up

Export Citation Format

Share Document