scholarly journals gapFinisher: a reliable gap filling pipeline for SSPACE-LongRead scaffolder output

2017 ◽  
Author(s):  
Juhana I Kammonen ◽  
Olli-Pekka Smolander ◽  
Lars Paulin ◽  
Pedro AB Pereira ◽  
Pia Laine ◽  
...  
Keyword(s):  
De Novo ◽  
Draft Genome ◽  
Sequence Information ◽  
Sequencing Data ◽  
Gap Filling ◽  
Third Generation Sequencing ◽  
Long Reads ◽  
Unix Command ◽  
Sequencing Platforms ◽  
Scaffolding Software

Unknown sequences, or gaps, are largely present in most published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while many computational tools exist partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding software that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the actual scaffolding. gapFinisher is based on controlled use of a gap filling tool called FGAP and works on all standard Linux/UNIX command lines. We conclude that performing the workflows of SSPACE-LongRead and gapFinisher enables users to fill gaps reliably. There is no need for further scrutiny of the existing sequencing data after performing the analysis.

scholarly journals gapFinisher: a reliable gap filling pipeline for SSPACE-LongRead scaffolder output

2017 ◽  
Author(s):  
Juhana I Kammonen ◽  
Olli-Pekka Smolander ◽  
Lars Paulin ◽  
Pedro AB Pereira ◽  
Pia Laine ◽  
...  
Keyword(s):  
De Novo ◽  
Draft Genome ◽  
Sequence Information ◽  
Sequencing Data ◽  
Gap Filling ◽  
Third Generation Sequencing ◽  
Long Reads ◽  
Unix Command ◽  
Sequencing Platforms ◽  
Scaffolding Software

Unknown sequences, or gaps, are largely present in most published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while many computational tools exist partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding software that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the actual scaffolding. gapFinisher is based on controlled use of a gap filling tool called FGAP and works on all standard Linux/UNIX command lines. We conclude that performing the workflows of SSPACE-LongRead and gapFinisher enables users to fill gaps reliably. There is no need for further scrutiny of the existing sequencing data after performing the analysis.

IsoDetect: Detection of splice isoforms from third generation long reads based on short feature sequences

2020 ◽  
Vol 15 ◽  
Author(s):  
Hongdong Li ◽  
Wenjing Zhang ◽  
Yuwen Luo ◽  
Jianxin Wang
Keyword(s):  
Sequence Similarity ◽  
Detection Methods ◽  
Sequence Information ◽  
Third Generation ◽  
Sequencing Data ◽  
Splice Isoforms ◽  
Third Generation Sequencing ◽  
Long Reads ◽  
Feature Sequence ◽  
Generation Sequencing

Aims: Accurately detect isoforms from third generation sequencing data. Background: Transcriptome annotation is the basis for the analysis of gene expression and regulation. The transcriptome annotation of many organisms such as humans is far from incomplete, due partly to the challenge in the identification of isoforms that are produced from the same gene through alternative splicing. Third generation sequencing (TGS) reads provide unprecedented opportunity for detecting isoforms due to their long length that exceeds the length of most isoforms. One limitation of current TGS reads-based isoform detection methods is that they are exclusively based on sequence reads, without incorporating the sequence information of known isoforms. Objective: Develop an efficient method for isoform detection. Method: Based on annotated isoforms, we propose a splice isoform detection method called IsoDetect. First, the sequence at exon-exon junction is extracted from annotated isoforms as the “short feature sequence”, which is used to distinguish different splice isoforms. Second, we aligned these feature sequences to long reads and divided long reads into groups that contain the same set of feature sequences, thereby avoiding the pair-wise comparison among the large number of long reads. Third, clustering and consensus generation are carried out based on sequence similarity. For the long reads that do not contain any short feature sequence, clustering analysis based on sequence similarity is performed to identify isoforms. Result: Tested on two datasets from Calypte Anna and Zebra Finch, IsoDetect showed higher speed and compelling accuracy compared with four existing methods. Conclusion: IsoDetect is a promising method for isoform detection. Other: This paper was accepted by the CBC2019 conference.

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 Strainline: full-length de novo viral haplotype reconstruction from noisy long reads

2021 ◽  
Author(s):  
Xiao Luo ◽  
Xiongbin Kang ◽  
Alexander Schoenhuth
Keyword(s):  
De Novo ◽  
Consensus Sequence ◽  
Full Length ◽  
Sequencing Data ◽  
Third Generation Sequencing ◽  
Novel Approach ◽  
Long Reads ◽  
Virus Genomes ◽  
Generation Sequencing ◽  
Diverse Virus

Haplotype-resolved de novo assembly of highly diverse virus genomes is critical in prevention, control and treatment of viral diseases. Current methods either can handle only relatively accurate short read data, or collapse haplotype-specific variations into consensus sequence. Here, we present Strainline, a novel approach to assemble viral haplotypes from noisy long reads without a reference genome. As a crucial consequence, Strainline is the first approach to provide strain-resolved, full-length de novo assemblies of viral quasispecies from noisy third-generation sequencing data. Benchmarking experiments on both simulated and real datasets of varying complexity and diversity confirm this novelty, by demonstrating the superiority of Strainline in terms of relevant criteria in comparison with the state of the art.

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 TrancriptomeReconstructoR, A Data-Driven Annotation of Complex Transcriptomes

2020 ◽  
Author(s):  
Maxim Ivanov ◽  
Albin Sandelin ◽  
Sebastian Marquardt
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
R Package ◽  
Sequence Information ◽  
Rna Seq ◽  
Sequencing Data ◽  
Gene Model ◽  
Preparation Methods ◽  
Downstream Analysis

Abstract Background: The quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data. Results: We developed TranscriptomeReconstructoR, an R package which implements a pipeline for automated transcriptome annotation. It relies on integrating features from independent and complementary datasets: i) full-length RNA-seq for detection of splicing patterns and ii) high-throughput 5' and 3' tag sequencing data for accurate definition of gene borders. The pipeline can also take a nascent RNA-seq dataset to supplement the called gene model with transient transcripts.We reconstructed de novo the transcriptional landscape of wild type Arabidopsis thaliana seedlings as a proof-of-principle. A comparison to the existing transcriptome annotations revealed that our gene model is more accurate and comprehensive than the two most commonly used community gene models, TAIR10 and Araport11. In particular, we identify thousands of transient transcripts missing from the existing annotations. Our new annotation promises to improve the quality of A.thaliana genome research.Conclusions: Our proof-of-concept data suggest a cost-efficient strategy for rapid and accurate annotation of complex eukaryotic transcriptomes. We combine the choice of library preparation methods and sequencing platforms with the dedicated computational pipeline implemented in the TranscriptomeReconstructoR package. The pipeline only requires prior knowledge on the reference genomic DNA sequence, but not the transcriptome. The package seamlessly integrates with Bioconductor packages for downstream analysis.

Computational Approaches for Transcriptome Assembly Based on Sequencing Technologies

2020 ◽  
Vol 15 (1) ◽  
pp. 2-16
Author(s):  
Yuwen Luo ◽  
Xingyu Liao ◽  
Fang-Xiang Wu ◽  
Jianxin Wang
Keyword(s):  
De Novo ◽  
Transcriptome Assembly ◽  
Critical Role ◽  
High Sensitivity ◽  
Biological Properties ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Massive Sequencing ◽  
Generation Sequencing

Transcriptome assembly plays a critical role in studying biological properties and examining the expression levels of genomes in specific cells. It is also the basis of many downstream analyses. With the increase of speed and the decrease in cost, massive sequencing data continues to accumulate. A large number of assembly strategies based on different computational methods and experiments have been developed. How to efficiently perform transcriptome assembly with high sensitivity and accuracy becomes a key issue. In this work, the issues with transcriptome assembly are explored based on different sequencing technologies. Specifically, transcriptome assemblies with next-generation sequencing reads are divided into reference-based assemblies and de novo assemblies. The examples of different species are used to illustrate that long reads produced by the third-generation sequencing technologies can cover fulllength transcripts without assemblies. In addition, different transcriptome assemblies using the Hybrid-seq methods and other tools are also summarized. Finally, we discuss the future directions of transcriptome assemblies.

scholarly journals NanoGalaxy: Nanopore long-read sequencing data analysis in Galaxy

GigaScience ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Willem de Koning ◽  
Milad Miladi ◽  
Saskia Hiltemann ◽  
Astrid Heikema ◽  
John P Hays ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Bioinformatics Analysis ◽  
De Novo ◽  
Sequence Data ◽  
Ease Of Use ◽  
Easy Access ◽  
Complex Data ◽  
Sequencing Data ◽  
Long Read ◽  
Sequencing Platforms

Abstract Background Long-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers. Results The Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads. Conclusions A range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

scholarly journals A benchmark of structural variation detection by long reads through a realistic simulated model

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nicolas Dierckxsens ◽  
Tong Li ◽  
Joris R. Vermeesch ◽  
Zhi Xie
Keyword(s):  
Structural Variation ◽  
New Method ◽  
Sequencing Data ◽  
Simulated Model ◽  
Long Reads ◽  
Long Read ◽  
Sequencing Platforms

AbstractAccurate simulations of structural variation distributions and sequencing data are crucial for the development and benchmarking of new tools. We develop Sim-it, a straightforward tool for the simulation of both structural variation and long-read data. These simulations from Sim-it reveal the strengths and weaknesses for current available structural variation callers and long-read sequencing platforms. With these findings, we develop a new method (combiSV) that can combine the results from structural variation callers into a superior call set with increased recall and precision, which is also observed for the latest structural variation benchmark set developed by the GIAB Consortium.

scholarly journals Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China

2016 ◽  
Author(s):  
Ying Wang ◽  
Kun Liu ◽  
De Bi ◽  
Biao Shou Zhou ◽  
Wen Jian Shao
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
Sequence Similarity ◽  
Molecular Study ◽  
Sequence Information ◽  
Sequencing Data ◽  
Protein Database ◽  
Illumina Hiseq ◽  
Significant Similarity ◽  
Assembly Technology

Background. Resurrection plants constitute a unique cadre within angiosperms. Boea clarkeana Hemsl. (Boea, Gesneriaceae) is a desiccation-tolerant dicotyledonous herb that is endemic to China. Although research on angiosperms with DT could be instructive for crops, genomic resources for B. clarkeana remain scarce. In addition, transcriptome sequencing could be an effective way to study desiccation-tolerant plants. Methods. In the present study, we used the platform Illumina HiSeqTM 2000 and de novo assembly technology to obtain leaf transcriptomes of B. clarkeana and conducted a BLASTX alignment of the sequencing data and protein databases for sequence classification and annotation. Then, based on the sequence information obtained, we developed EST-SSR markers by means of EST-SSR mining, primer design and polymorphism identification. Results. A total of 91,449 unigenes were generated from the leaf cDNA library of B. clarkeana in this study. Based on a sequence similarity search with a known protein database, 72,087 unigenes were annotated. Among the annotated unigenes, a total of 71,170 unigenes showed significant similarity to known proteins of 463 popular model species in the Nr database, and 59,962 unigenes and 32,336 unigenes were assigned to GO classifications and COG, respectively. In addition, 44,924 unigenes were mapped in 128 KEGG pathways. Furthermore, a total of 7,610 unigenes with 8,563 microsatellites were found. Seventy-four primer pairs were selected from 436 primer pairs designed for polymorphism validation. SSRs with higher polymorphism rates were concentrated on dinucleotides, pentanucleotides and hexanucleotides. Finally, 17 pairs with highly polymorphic and stable loci were selected for polymorphism screening. There were a total of 65 alleles, with 2–6 alleles at each locus. Mainly due to the unique biological characteristics of plants, the HE, HO and PIC per locus were very low, ranging from 0 to 0.196, 0.082 to 0.14 and 0 to 0.155, respectively. Discussion. A substantial fraction transcriptome sequences of B. clarkeana were generated in this study, which is the first molecular-level analysis of this plant. These sequences are valuable resources for gene annotation and discovery and molecular marker development. These sequences could also provide a valuable basis for the future molecular study of B. clarkeana.

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