scholarly journals Strain-aware assembly of genomes from mixed samples using flow variation graphs

2019 ◽  
Author(s):  
Jasmijn A. Baaijens ◽  
Leen Stougie ◽  
Alexander Schönhuth
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Current Approach ◽  
Full Length ◽  
Strain Level ◽  
Abundance Estimation ◽  
Flow Variation ◽  
Assembly Accuracy ◽  
Mixed Samples

AbstractThe goal of strain-aware genome assembly is to reconstruct all individual haplotypes from a mixed sample at the strain level and to provide abundance estimates for the strains. Given that the use of a reference genome can introduce significant biases, de novo approaches are most suitable for this task. So far, reference-genome-independent assemblers have been shown to reconstruct haplotypes for mixed samples of limited complexity and genomes not exceeding 10000 bp in length.Here, we present VG-Flow, a de novo approach that enables full-length haplotype reconstruction from pre-assembled contigs of complex mixed samples. Our method increases contiguity of the input assembly and, at the same time, it performs haplotype abundance estimation. VG-Flow is the first approach to require polynomial, and not exponential runtime in terms of the underlying graphs. Since runtime increases only linearly in the length of the genomes in practice, it enables the reconstruction also of genomes that are longer by orders of magnitude, thereby establishing the first de novo solution to strain-aware full-length genome assembly applicable to bacterial sized genomes.VG-Flow is based on the flow variation graph as a novel concept that both captures all diversity present in the sample and enables to cast the central contig abundance estimation problem as a flow-like, polynomial time solvable optimization problem. As a consequence, we are in position to compute maximal-length haplotypes in terms of decomposing the resulting flow efficiently using a greedy algorithm, and obtain accurate frequency estimates for the reconstructed haplotypes through linear programming techniques.Benchmarking experiments show that our method outperforms state-of-the-art approaches on mixed samples from short genomes in terms of assembly accuracy as well as abundance estimation. Experiments on longer, bacterial sized genomes demonstrate that VG-Flow is the only current approach that can reconstruct full-length haplotypes from mixed samples at the strain level in human-affordable runtime.

scholarly journals Genome assembly of the JD17 soybean provides a new reference genome for Comparative genomics

2021 ◽  
Author(s):  
Xinxin Yi ◽  
Jing Liu ◽  
Shengcai Chen ◽  
Hao Wu ◽  
Min Liu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
High Quality ◽  
Genome Wide ◽  
A Genome ◽  
Cultivated Soybean

Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromsome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with three published soybeans (WM82, ZH13 and W05) , which identified five large inversions and two large translocations specific to JD17, 20,984 - 46,912 PAVs spanning 13.1 - 46.9 Mb in size, and 5 - 53 large PAV clusters larger than 500kb. 1,695,741 - 3,664,629 SNPs and 446,689 - 800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation (SNF) genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.

scholarly journals Highly Contiguous Nanopore Genome Assembly of Chlamydomonas reinhardtii CC-1690

2020 ◽  
Vol 9 (37) ◽  
Author(s):  
Samuel O’Donnell ◽  
Frederic Chaux ◽  
Gilles Fischer
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Genome Size ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Content Type ◽  
Oxford Nanopore ◽  
A Genome ◽  
Reference Quality

ABSTRACT The current Chlamydomonas reinhardtii reference genome remains fragmented due to gaps stemming from large repetitive regions. To overcome the vast majority of these gaps, publicly available Oxford Nanopore Technology data were used to create a new reference-quality de novo genome assembly containing only 21 contigs, 30/34 telomeric ends, and a genome size of 111 Mb.

scholarly journals dnAQET: a framework to compute a consolidated metric for benchmarking quality of de novo assemblies

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Gokhan Yavas ◽  
Huixiao Hong ◽  
Wenming Xiao
Keyword(s):  
Quality Assessment ◽  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Quality Score ◽  
De Novo Genome Assembly ◽  
Genome Assemblies ◽  
Reference Genomes ◽  
Better Than

Abstract Background Accurate de novo genome assembly has become reality with the advancements in sequencing technology. With the ever-increasing number of de novo genome assembly tools, assessing the quality of assemblies has become of great importance in genome research. Although many quality metrics have been proposed and software tools for calculating those metrics have been developed, the existing tools do not produce a unified measure to reflect the overall quality of an assembly. Results To address this issue, we developed the de novo Assembly Quality Evaluation Tool (dnAQET) that generates a unified metric for benchmarking the quality assessment of assemblies. Our framework first calculates individual quality scores for the scaffolds/contigs of an assembly by aligning them to a reference genome. Next, it computes a quality score for the assembly using its overall reference genome coverage, the quality score distribution of its scaffolds and the redundancy identified in it. Using synthetic assemblies randomly generated from the latest human genome build, various builds of the reference genomes for five organisms and six de novo assemblies for sample NA24385, we tested dnAQET to assess its capability for benchmarking quality evaluation of genome assemblies. For synthetic data, our quality score increased with decreasing number of misassemblies and redundancy and increasing average contig length and coverage, as expected. For genome builds, dnAQET quality score calculated for a more recent reference genome was better than the score for an older version. To compare with some of the most frequently used measures, 13 other quality measures were calculated. The quality score from dnAQET was found to be better than all other measures in terms of consistency with the known quality of the reference genomes, indicating that dnAQET is reliable for benchmarking quality assessment of de novo genome assemblies. Conclusions The dnAQET is a scalable framework designed to evaluate a de novo genome assembly based on the aggregated quality of its scaffolds (or contigs). Our results demonstrated that dnAQET quality score is reliable for benchmarking quality assessment of genome assemblies. The dnQAET can help researchers to identify the most suitable assembly tools and to select high quality assemblies generated.

scholarly journals De novo assembly of the cattle reference genome with single-molecule sequencing

GigaScience ◽  
2020 ◽  
Vol 9 (3) ◽  
Author(s):  
Benjamin D Rosen ◽  
Derek M Bickhart ◽  
Robert D Schnabel ◽  
Sergey Koren ◽  
Christine G Elsik ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Bos Taurus ◽  
Future Research ◽  
Protein Coding ◽  
Single Molecule Sequencing ◽  
Assembly Accuracy ◽  
Genomic Tools

Abstract Background Major advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10–12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. Results We present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250× more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use. Conclusions We demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species.

42 An Improved, High-quality Ovine Reference Genome Assembly

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 23-24
Author(s):  
Kimberly M Davenport ◽  
Derek M Bickhart ◽  
Kim Worley ◽  
Shwetha C Murali ◽  
Noelle Cockett ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Functional Annotation ◽  
Reference Genome ◽  
De Novo ◽  
The United States ◽  
Read Length ◽  
Chromosome 11 ◽  
High Quality ◽  
Oxford Nanopore ◽  
Long Read

Abstract Sheep are an important agricultural species used for both food and fiber in the United States and globally. A high-quality reference genome enhances the ability to discover genetic and biological mechanisms influencing important traits, such as meat and wool quality. The rapid advances in genome assembly algorithms and emergence of increasingly long sequence read length provide the opportunity for an improved de novo assembly of the sheep reference genome. Tissue was collected postmortem from an adult Rambouillet ewe selected by USDA-ARS for the Ovine Functional Annotation of Animal Genomes project. Short-read (55x coverage), long-read PacBio (75x coverage), and Hi-C data from this ewe were retrieved from public databases. We generated an additional 50x coverage of Oxford Nanopore data and assembled the combined long-read data with canu v1.9. The assembled contigs were polished with Nanopolish v0.12.5 and scaffolded using Hi-C data with Salsa v2.2. Gaps were filled with PBsuite v15.8.24 and polished with Nanopolish v0.12.5 followed by removal of duplicate contigs with PurgeDups v1.0.1. Chromosomes were oriented by identifying centromeres and telomeres with RepeatMasker v4.1.1, indicating a need to reverse the orientation of chromosome 11 relative to Oar_rambouillet_v1.0. Final polishing was performed with two rounds of a pipeline which consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly has improved continuity (contig N50 of 43.19 Mb) with a 19-fold and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. This significantly improved reference assembly, public at NCBI GenBank under accession number GCA_016772045, will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits relevant the sheep industry.

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 Full-length de novo viral quasispecies assembly through variation graph construction

2018 ◽  
Author(s):  
Jasmijn A. Baaijens ◽  
Bastiaan Van der Roest ◽  
Johannes Köster ◽  
Leen Stougie ◽  
Alexander Schönhuth
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Simulated Data ◽  
Error Rates ◽  
Full Length ◽  
Maximal Length ◽  
Data Sets ◽  
Viral Quasispecies ◽  
Suitable Structure ◽  
Selection Of

AbstractMotivationViruses populate their hosts as a viral quasispecies: a collection of genetically related mutant strains. Viral quasispecies assembly refers to reconstructing the strain-specific haplotypes from read data, and predicting their relative abundances within the mix of strains, an important step for various treatment-related reasons. Reference-genome-independent (“de novo”) approaches have yielded benefits over reference-guided approaches, because reference-induced biases can become overwhelming when dealing with divergent strains. While being very accurate, extant de novo methods only yield rather short contigs. It remains to reconstruct full-length haplotypes together with their abundances from such contigs.MethodWe first construct a variation graph, a recently popular, suitable structure for arranging and integrating several related genomes, from the short input contigs, without making use of a reference genome. To obtain paths through the variation graph that reflect the original haplotypes, we solve a minimization problem that yields a selection of maximal-length paths that is optimal in terms of being compatible with the read coverages computed for the nodes of the variation graph. We output the resulting selection of maximal length paths as the haplotypes, together with their abundances.ResultsBenchmarking experiments on challenging simulated data sets show significant improvements in assembly contiguity compared to the input contigs, while preserving low error rates. As a consequence, our method outperforms all state-of-the-art viral quasispecies assemblers that aim at the construction of full-length haplotypes, in terms of various relevant assembly measures. Our tool, Virus-VG, is publicly available at https://bitbucket.org/jbaaijens/virus-vg.

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.

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 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.

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