scholarly journals Jasmine: Population-scale structural variant comparison and analysis

2021 ◽  
Author(s):  
Melanie Kirsche ◽  
Gautam Prabhu ◽  
Rachel Sherman ◽  
Bohan Ni ◽  
Sergey Aganezov ◽  
...  
Keyword(s):  
De Novo ◽  
Population Analysis ◽  
Accurate Method ◽  
Structural Variants ◽  
Sequencing Data ◽  
1000 Genomes ◽  
Dna And Rna ◽  
Long Reads ◽  
Long Read ◽  
Proximity Graph

The increasing availability of long-reads is revolutionizing studies of structural variants (SVs). However, because SVs vary across individuals and are discovered through imprecise read technologies and methods, they can be difficult to compare. Addressing this, we present Jasmine (https://github.com/mkirsche/Jasmine), a fast and accurate method for SV refinement, comparison, and population analysis. Using an SV proximity graph, Jasmine outperforms five widely-used comparison methods, including reducing the rate of Mendelian discordance in trio datasets by more than five-fold, and reveals a set of high confidence de novo SVs confirmed by multiple long-read technologies. We also present a harmonized callset of 205,192 SVs from 31 samples of diverse ancestry sequenced with long reads. We genotype these SVs in 444 short read samples from the 1000 Genomes Project with both DNA and RNA sequencing data and assess their widespread impact on gene expression, including within several medically relevant genes.

scholarly journals De novo assembly of a Tibetan genome and identification of novel structural variants associated with high-altitude adaptation

2019 ◽  
Vol 7 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Yaoxi He ◽  
Haiyi Lou ◽  
Chaoying Cui ◽  
Lian Deng ◽  
Yang Gao ◽  
...  
Keyword(s):  
High Altitude ◽  
De Novo Assembly ◽  
De Novo ◽  
Population Analysis ◽  
Extreme Environments ◽  
Structural Variants ◽  
Base Pairs ◽  
High Quality ◽  
Long Read ◽  
Altitude Adaptation

Abstract Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.

scholarly journals Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xin Luo ◽  
Yaoxi He ◽  
Chao Zhang ◽  
Xiechao He ◽  
Lanzhen Yan ◽  
...  
Keyword(s):  
Rhesus Monkeys ◽  
De Novo ◽  
Preclinical Model ◽  
Structural Variants ◽  
Sequencing Data ◽  
De Novo Mutations ◽  
Target Region ◽  
Long Read ◽  
Target Effect

AbstractCRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.

scholarly journals Clustering de Novo by Gene of Long Reads from Transcriptomics Data

2017 ◽  
Author(s):  
Camille Marchet ◽  
Lolita Lecompte ◽  
Corinne Da Silva ◽  
Corinne Cruaud ◽  
Jean-Marc Aury ◽  
...  
Keyword(s):  
De Novo ◽  
Free Access ◽  
Sequencing Data ◽  
Base Pairs ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Processing Step ◽  
Whole Transcriptome Sequencing ◽  
Long Read ◽  
Transcriptomics Data

AbstractLong-read sequencing currently provides sequences of several thousand base pairs. This allows to obtain complete transcripts, which offers an un-precedented vision of the cellular transcriptome.However the literature is lacking tools to cluster such data de novo, in particular for Oxford Nanopore Technologies reads, because of the inherent high error rate compared to short reads.Our goal is to process reads from whole transcriptome sequencing data accurately and without a reference genome in order to reliably group reads coming from the same gene. This de novo approach is therefore particularly suitable for non-model species, but can also serve as a useful pre-processing step to improve read mapping. Our contribution is both to propose a new algorithm adapted to clustering of reads by gene and a practical and free access tool that permits to scale the complete processing of eukaryotic transcriptomes.We sequenced a mouse RNA sample using the MinION device, this dataset is used to compare our solution to other algorithms used in the context of biological clustering. We demonstrate its is better-suited for transcriptomics long reads. When a reference is available thus mapping possible, we show that it stands as an alternative method that predicts complementary clusters.

scholarly journals Accurate Haplotype-Resolved Assembly Reveals The Origin Of Structural Variants For Human Trios

2021 ◽  
Author(s):  
Mengyang Xu ◽  
Lidong Guo ◽  
Xiao Du ◽  
Lei Li ◽  
Brock A Peters ◽  
...  
Keyword(s):  
De Novo ◽  
Substantial Improvement ◽  
Supplementary Information ◽  
Sequencing Data ◽  
Homologous Chromosomes ◽  
Assembly Method ◽  
Long Reads ◽  
Long Read ◽  
Second Generation Sequencing ◽  
Generation Sequencing

Abstract Motivation Achieving a near complete understanding of how the genome of an individual affects the phenotypes of that individual requires deciphering the order of variations along homologous chromosomes in species with diploid genomes. However, true diploid assembly of long-range haplotypes remains challenging. Results To address this, we have developed Haplotype-resolved Assembly for Synthetic long reads using a Trio-binning strategy, or HAST, which uses parental information to classify reads into maternal or paternal. Once sorted, these reads are used to independently de novo assemble the parent-specific haplotypes. We applied HAST to co-barcoded second-generation sequencing data from an Asian individual, resulting in a haplotype assembly covering 94.7% of the reference genome with a scaffold N50 longer than 11 Mb. The high haplotyping precision (∼99.7%) and recall (∼95.9%) represents a substantial improvement over the commonly used tool for assembling co-barcoded reads (Supernova), and is comparable to a trio-binning-based third generation long-read based assembly method (TrioCanu) but with a significantly higher single-base accuracy (up to 99.99997% (Q65)). This makes HAST a superior tool for accurate haplotyping and future haplotype-based studies. Availability The code of the analysis is available at https://github.com/BGI-Qingdao/HAST. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

2019 ◽  
Author(s):  
Aaron M. Wenger ◽  
Paul Peluso ◽  
William J. Rowell ◽  
Pi-Chuan Chang ◽  
Richard J. Hall ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Variant Detection ◽  
High Quality Genome ◽  
Circular Consensus Sequencing

AbstractThe major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

scholarly journals Robust Benchmark Structural Variant Calls of An Asian Using the State-of-Art Long Fragment Sequencing Technologies

2020 ◽  
Author(s):  
Xiao Du ◽  
Lili Li ◽  
Fan Liang ◽  
Sanyang Liu ◽  
Wenxin Zhang ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
De Novo ◽  
Structural Variants ◽  
High Confidence ◽  
False Negatives ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Circular Consensus Sequencing

AbstractThe importance of structural variants (SVs) on phenotypes and human diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of those approaches, our work established an Asian reference material comprising identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8,938 SVs in an EBV immortalized B lymphocyte line, by integrating four alignment-based SV callers [from 109× PacBio continuous long read (CLR), 22× PacBio circular consensus sequencing (CCS) reads, 104× Oxford Nanopore long reads, and 114× optical mapping platform (Bionano)] and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR and Sanger sequencing, proofing the robustness of our SV calls. Combining trio-binning based haplotype assemblies, we established an SV benchmark for identification of false negatives and false positives by constructing the continuous high confident regions (CHCRs), which cover 1.46Gb and 6,882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical diagnosis.

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 WENGAN: Efficient and high quality hybrid de novo assembly of human genomes

2019 ◽  
Author(s):  
Alex Di Genova ◽  
Elena Buena-Atienza ◽  
Stephan Ossowski ◽  
Marie-France Sagot
Keyword(s):  
De Novo ◽  
Computational Cost ◽  
Sequence Information ◽  
Sequencing Data ◽  
High Quality ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Long Reads ◽  
Long Read ◽  
Genome Assemblies

The continuous improvement of long-read sequencing technologies along with the development of ad-doc algorithms has launched a new de novo assembly era that promises high-quality genomes. However, it has proven difficult to use only long reads to generate accurate genome assemblies of large, repeat-rich human genomes. To date, most of the human genomes assembled from long error-prone reads add accurate short reads to further polish the consensus quality. Here, we report the development of a novel algorithm for hybrid assembly, WENGAN, and the de novo assembly of four human genomes using a combination of sequencing data generated on ONT PromethION, PacBio Sequel, Illumina and MGI technology. WENGAN implements efficient algorithms that exploit the sequence information of short and long reads to tackle assembly contiguity as well as consensus quality. The resulting genome assemblies have high contiguity (contig NG50:16.67-62.06 Mb), few assembly errors (contig NGA50:10.9-45.91 Mb), good consensus quality (QV:27.79-33.61), and high gene completeness (BUSCO complete: 94.6-95.1%), while consuming low computational resources (CPU hours:153-1027). In particular, the WENGAN assembly of the haploid CHM13 sample achieved a contig NG50 of 62.06 Mb (NGA50:45.91 Mb), which surpasses the contiguity of the current human reference genome (GRCh38 contig NG50:57.88 Mb). Providing highest quality at low computational cost, WENGAN is an important step towards the democratization of the de novo assembly of human genomes. The WENGAN assembler is available at https://github.com/adigenova/wengan

scholarly journals High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Nick Vereecke ◽  
Jade Bokma ◽  
Freddy Haesebrouck ◽  
Hans Nauwynck ◽  
Filip Boyen ◽  
...  
Keyword(s):  
De Novo ◽  
Respiratory Pathogen ◽  
Mycoplasma Bovis ◽  
Sequencing Data ◽  
Molecular Approaches ◽  
Long Reads ◽  
Long Read ◽  
Control And Prevention ◽  
Single Use ◽  
Cost Efficient

Abstract Background Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1–2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained. Results Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness). Conclusions These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.

scholarly journals Benchmarking metagenomic classification tools for long-read sequencing data

2020 ◽  
Author(s):  
Josip Marić ◽  
Krešimir Križanović ◽  
Sylvain Riondet ◽  
Niranjan Nagarajan ◽  
Mile Šikić
Keyword(s):  
De Novo ◽  
Real Life ◽  
Metagenomic Analysis ◽  
Sequencing Data ◽  
De Novo Genome Assembly ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Species Abundances ◽  
Long Read ◽  
Eukaryotic Genomes

ABSTRACTIn recent years, both long-read sequencing and metagenomic analysis have been significantly advanced. Although long-read sequencing technologies have been primarily used for de novo genome assembly, they are rapidly maturing for widespread use in other applications. In particular, long reads could potentially lead to more precise taxonomic identification, which has sparked an interest in using them for metagenomic analysis.Here we present a benchmark of several state-of-the-art tools for metagenomic taxonomic classification, tested on in-silico datasets constructed using real long reads from isolate sequencing. We compare tools that were either newly developed or modified to work with long reads, including k-mer based tools Kraken2, Centrifuge and CLARK, and mapping-based tools MetaMaps and MEGAN-LR. The test datasets were constructed with varying numbers of bacterial and eukaryotic genomes to simulate different real-life metagenomic applications. The tools were tested to detect species accurately and precisely estimate species abundances in the samples.Our analysis shows that all tested classifiers provide useful results, and the composition of the used database strongly influences the performance. Using the same database, tested tools achieve comparable results except for MetaMaps, which slightly outperform others in most metrics, but it is significantly slower than k-mer based tools.We deem there is significant room for improvement for all tested tools, especially in lowering the number of false-positive detections.

Sign in / Sign up

Export Citation Format

Share Document