scholarly journals Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 481 ◽  
Author(s):  
Chen ◽  
Lin ◽  
Xie ◽  
Zhong ◽  
Zhang ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Single Molecule ◽  
Functional Categories ◽  
Genetic Studies ◽  
Sequencing Technologies ◽  
Clusters Of Orthologous Groups ◽  
Long Read ◽  
Main Gene ◽  
First Time ◽  
Main Factor

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

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.

scholarly journals PacBio Single-Molecule Long-Read Sequencing Reveals Genes Tolerating Manganese Stress in Schima superba Saplings

2021 ◽  
Vol 12 ◽  
Author(s):  
Fiza Liaquat ◽  
Muhammad Farooq Hussain Munis ◽  
Samiah Arif ◽  
Urooj Haroon ◽  
Jianxin Shi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gene Annotation ◽  
Treated Group ◽  
Full Length ◽  
Open Reading Frames ◽  
Interacting Protein ◽  
Schima Superba ◽  
Long Read ◽  
First Time ◽  
Potential Tool

Schima superba (Theaceae) is a subtropical evergreen tree and is used widely for forest firebreaks and gardening. It is a plant that tolerates salt and typically accumulates elevated amounts of manganese in the leaves. With large ecological amplitude, this tree species grows quickly. Due to its substantial biomass, it has a great potential for soil remediation. To evaluate the thorough framework of the mRNA, we employed PacBio sequencing technology for the first time to generate S. Superba transcriptome. In this analysis, overall, 511,759 full length non-chimeric reads were acquired, and 163,834 high-quality full-length reads were obtained. Overall, 93,362 open reading frames were obtained, of which 78,255 were complete. In gene annotation analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Non-Redundant (Nr) databases were allocated 91,082, 71,839, 38,914, and 38,376 transcripts, respectively. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT), and Coding Non-Coding Index (CNCI) databases and observed 8,551, 9,174, 20,720, and 18,669 lncRNAs, respectively. Moreover, nine genes were randomly selected for the expression analysis, which showed the highest expression of Gene 6 (Na_Ca_ex gene), and CAX (CAX-interacting protein 4) was higher in manganese (Mn)-treated group. This work provided significant number of full-length transcripts and refined the annotation of the reference genome, which will ease advanced genetic analyses of S. superba.

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals ISOdb: A Comprehensive Database of Full-Length Isoforms Generated by Iso-Seq

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Shang-Qian Xie ◽  
Yue Han ◽  
Xiao-Zhou Chen ◽  
Tai-Yu Cao ◽  
Kai-Kai Ji ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Public Access ◽  
Transcript Isoforms ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Depth Analysis ◽  
Gene Level ◽  
Long Read ◽  
Full Length Transcript

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

scholarly journals Quantifying the Benefit Offered by Transcript Assembly on Single-Molecule Long Reads

2019 ◽  
Author(s):  
Laura H. Tung ◽  
Mingfu Shao ◽  
Carl Kingsford
Keyword(s):  
Single Molecule ◽  
Error Rates ◽  
Human Transcriptome ◽  
Sequencing Technologies ◽  
Third Generation Sequencing ◽  
Long Reads ◽  
Long Read ◽  
Transcript Assembly ◽  
Novel Isoforms ◽  
Generation Sequencing

AbstractThird-generation sequencing technologies benefit transcriptome analysis by generating longer sequencing reads. However, not all single-molecule long reads represent full transcripts due to incomplete cDNA synthesis and the sequencing length limit of the platform. This drives a need for long read transcript assembly. We quantify the benefit that can be achieved by using a transcript assembler on long reads. Adding long-read-specific algorithms, we evolved Scallop to make Scallop-LR, a long-read transcript assembler, to handle the computational challenges arising from long read lengths and high error rates. Analyzing 26 SRA PacBio datasets using Scallop-LR, Iso-Seq Analysis, and StringTie, we quantified the amount by which assembly improved Iso-Seq results. Through combined evaluation methods, we found that Scallop-LR identifies 2100–4000 more (for 18 human datasets) or 1100–2200 more (for eight mouse datasets) known transcripts than Iso-Seq Analysis, which does not do assembly. Further, Scallop-LR finds 2.4–4.4 times more potentially novel isoforms than Iso-Seq Analysis for the human and mouse datasets. StringTie also identifies more transcripts than Iso-Seq Analysis. Adding long-read-specific optimizations in Scallop-LR increases the numbers of predicted known transcripts and potentially novel isoforms for the human transcriptome compared to several recent short-read assemblers (e.g. StringTie). Our findings indicate that transcript assembly by Scallop-LR can reveal a more complete human transcriptome.

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 Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

2021 ◽  
Author(s):  
Leho Tedersoo ◽  
Mads Albertsen ◽  
Sten Anslan ◽  
Benjamin Callahan
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

scholarly journals Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Peter Edge ◽  
Vikas Bansal
Keyword(s):  
Single Molecule ◽  
Variant Calling ◽  
Small Scale ◽  
Whole Genome ◽  
Limited Information ◽  
Single Nucleotide Variants ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

Abstract Whole-genome sequencing using sequencing technologies such as Illumina enables the accurate detection of small-scale variants but provides limited information about haplotypes and variants in repetitive regions of the human genome. Single-molecule sequencing (SMS) technologies such as Pacific Biosciences and Oxford Nanopore generate long reads that can potentially address the limitations of short-read sequencing. However, the high error rate of SMS reads makes it challenging to detect small-scale variants in diploid genomes. We introduce a variant calling method, Longshot, which leverages the haplotype information present in SMS reads to accurately detect and phase single-nucleotide variants (SNVs) in diploid genomes. We demonstrate that Longshot achieves very high accuracy for SNV detection using whole-genome Pacific Biosciences data, outperforms existing variant calling methods, and enables variant detection in duplicated regions of the genome that cannot be mapped using short reads.

scholarly journals Hapo-G, Haplotype-Aware Polishing Of Genome Assemblies

2020 ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
Short Reads ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

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 short reads to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

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