scholarly journals Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

2018 ◽  
Author(s):  
Huilong Du ◽  
Chengzhi Liang
Keyword(s):  
Single Molecule ◽  
High Efficiency ◽  
Reference Genome ◽  
Repetitive Sequences ◽  
Sequencing Data ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Genome Maps ◽  
Long Reads ◽  
Novel Method

AbstractDue to the large number of repetitive sequences in complex eukaryotic genomes, fragmented and incompletely assembled genomes lose value as reference sequences, often due to short contigs that cannot be anchored or mispositioned onto chromosomes. Here we report a novel method Highly Efficient Repeat Assembly (HERA), which includes a new concept called a connection graph as well as algorithms for constructing the graph. HERA resolves repeats at high efficiency with single-molecule sequencing data, and enables the assembly of chromosome-scale contigs by further integrating genome maps and Hi-C data. We tested HERA with the genomes of rice R498, maize B73, human HX1 and Tartary buckwheat Pinku1. HERA can correctly assemble most of the tandemly repetitive sequences in rice using single-molecule sequencing data only. Using the same maize and human sequencing data published by Jiao et al. (2017) and Shi et al. (2016), respectively, we dramatically improved on the sequence contiguity compared with the published assemblies, increasing the contig N50 from 1.3 Mb to 61.2 Mb in maize B73 assembly and from 8.3 Mb to 54.4 Mb in human HX1 assembly with HERA. We provided a high-quality maize reference genome with 96.9% of the gaps filled (only 76 gaps left) and several incorrectly positioned sequences fixed compared with the B73 RefGen_v4 assembly. Comparisons between the HERA assembly of HX1 and the human GRCh38 reference genome showed that many gaps in GRCh38 could be filled, and that GRCh38 contained some potential errors that could be fixed. We assembled the Pinku1 genome into 12 scaffolds with a contig N50 size of 27.85 Mb. HERA serves as a new genome assembly/phasing method to generate high quality sequences for complex genomes and as a curation tool to improve the contiguity and completeness of existing reference genomes, including the correction of assembly errors in repetitive regions.

scholarly journals A high-quality reference genome for a parasitic bivalve with doubly uniparental inheritance (Bivalvia: Unionida)

2021 ◽  
Author(s):  
Chase H Smith
Keyword(s):  
Single Molecule ◽  
Reference Genome ◽  
Economic Value ◽  
Freshwater Mussel ◽  
Uniparental Inheritance ◽  
Doubly Uniparental Inheritance ◽  
High Quality ◽  
Long Reads ◽  
A Genome ◽  
Genomic Studies

Abstract From a genomics perspective, bivalves (Mollusca: Bivalvia) have been poorly explored with the exception for those of high economic value. The bivalve order Unionida, or freshwater mussels, has been of interest in recent genomic studies due to their unique mitochondrial biology and peculiar life cycle. However, genomic studies have been hindered by the lack of a high-quality reference genome. Here, I present a genome assembly of Potamilus streckersoni using Pacific Bioscience single-molecule real-time long reads and 10X Genomics linked read sequencing. Further, I use RNA sequencing from multiple tissue types and life stages to annotate the reference genome. The final assembly was far superior to any previously published freshwater mussel genome and was represented by 2,368 scaffolds (2,472 contigs) and 1,776,755,624 bp, with a scaffold N50 of 2,051,244 bp. A high proportion of the assembly was comprised of repetitive elements (51.03%), aligning with genomic characteristics of other bivalves. The functional annotation returned 52,407 gene models (41,065 protein, 11,342 tRNAs), which was concordant with the estimated number of genes in other freshwater mussel species. This genetic resource, along with future studies developing high-quality genome assemblies and annotations, will be integral toward unraveling the genomic bases of ecologically and evolutionarily important traits in this hyper-diverse group.

scholarly journals A chromosome-scale assembly of the major African malaria vector Anopheles funestus

2018 ◽  
Author(s):  
Jay Ghurye ◽  
Sergey Koren ◽  
Scott T Small ◽  
Seth Redmond ◽  
Paul Howell ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reference Genome ◽  
Anopheles Funestus ◽  
Genomic Variation ◽  
Phenotypic Traits ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Long Read ◽  
Haploid Genome Size ◽  
Important Disease

Background: Anopheles funestus is one of the three most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito. Findings: Here we present a new high-quality An. funestus reference genome (AfunF3) assembled using 240x coverage of long-read single-molecule sequencing for contigging, combined with 100x coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1. Conclusion: This highly contiguous and complete An. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector.

scholarly journals Genome resource for Elsinoë batatas, the causal agent of stem and foliage scab disease of sweet potato

2021 ◽  
Author(s):  
Xinxin Zhang ◽  
Hongda Zou ◽  
Yiling Yang ◽  
Boping Fang ◽  
Lifei Huang
Keyword(s):  
Sweet Potato ◽  
Single Molecule ◽  
Reference Genome ◽  
Gc Content ◽  
Basic Research ◽  
Phytopathogenic Fungus ◽  
Sequencing Technology ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
High Quality Genome

Elsinoë batatas is a phytopathogenic fungus causing stem and foliage scab disease of sweet potato. At present, there is no reference genome available for E. batatas, limiting basic research for the pathogen. The present study applied the nanopore single molecule sequencing technology to sequence the E. batatas genome. This study thus reports the first high-quality genome sequence of E. batatas, with a total contig size of 26.49 Mb, 50.8% GC content and an N50 of 2,546,814bp. The sequences obtained serve as a reference for analysis of E. batatas isolates and provide a resource to better understand the biology of stem and foliage scab disease of sweet potato.

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 SLR-superscaffolder: a de novo scaffolding tool for synthetic long reads using a top-to-bottom scheme

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lidong Guo ◽  
Mengyang Xu ◽  
Wenchao Wang ◽  
Shengqiang Gu ◽  
Xia Zhao ◽  
...  
Keyword(s):  
High Efficiency ◽  
De Novo ◽  
Next Generation Sequencing Data ◽  
Sequencing Data ◽  
Draft Assembly ◽  
Screening Algorithm ◽  
Long Reads ◽  
Hybrid Genome ◽  
Genomics Research ◽  
Negative Effect

Abstract Background Synthetic long reads (SLR) with long-range co-barcoding information are now widely applied in genomics research. Although several tools have been developed for each specific SLR technique, a robust standalone scaffolder with high efficiency is warranted for hybrid genome assembly. Results In this work, we developed a standalone scaffolding tool, SLR-superscaffolder, to link together contigs in draft assemblies using co-barcoding and paired-end read information. Our top-to-bottom scheme first builds a global scaffold graph based on Jaccard Similarity to determine the order and orientation of contigs, and then locally improves the scaffolds with the aid of paired-end information. We also exploited a screening algorithm to reduce the negative effect of misassembled contigs in the input assembly. We applied SLR-superscaffolder to a human single tube long fragment read sequencing dataset and increased the scaffold NG50 of its corresponding draft assembly 1349 fold. Moreover, benchmarking on different input contigs showed that this approach overall outperformed existing SLR scaffolders, providing longer contiguity and fewer misassemblies, especially for short contigs assembled by next-generation sequencing data. The open-source code of SLR-superscaffolder is available at https://github.com/BGI-Qingdao/SLR-superscaffolder. Conclusions SLR-superscaffolder can dramatically improve the contiguity of a draft assembly by integrating a hybrid assembly strategy.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

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 Genome assembly of the maize inbred line A188 provides a new reference genome for functional genomics

2021 ◽  
Author(s):  
Fei Ge ◽  
Jingtao Qu ◽  
Peng Liu ◽  
Lang Pan ◽  
Chaoying Zou ◽  
...  
Keyword(s):  
Single Molecule ◽  
Inbred Line ◽  
Genome Mapping ◽  
Maize Inbred Line ◽  
Sequencing Data ◽  
Structural Variations ◽  
Single Molecule Sequencing ◽  
Maize Genetic ◽  
Induction Ratio ◽  
Phenotypic Variations

Heretofore, little is known about the mechanism underlying the genotype-dependence of embryonic callus (EC) induction, which has severely inhibited the development of maize genetic engineering. Here, we report the genome sequence and annotation of a maize inbred line with high EC induction ratio, A188, which is assembled from single-molecule sequencing and optical genome mapping. We assembled a 2,210 Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to those of 9.73 Mb for B73 and 10.2 Mb for Mo17. Comparative analysis revealed that ~30% of the predicted A188 genes had large structural variations to B73, Mo17 and W22 genomes, which caused considerable protein divergence and might lead to phenotypic variations between the four inbred lines. Combining our new A188 genome, previously reported QTLs and RNA sequencing data, we reveal 8 large structural variation genes and 4 differentially expressed genes playing potential roles in EC induction.

scholarly journals De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joseph R. Fauver ◽  
John Martin ◽  
Gary J. Weil ◽  
Makedonka Mitreva ◽  
Peter U. Fischer
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Reference Genome ◽  
De Novo ◽  
Complete Mitochondrial Genome ◽  
Nuclear Genome ◽  
Brugia Malayi ◽  
Field Isolates ◽  
Sequencing Technologies ◽  
Long Reads

AbstractFilarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.

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