Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.

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Improving the Chromosome-Level Genome Assembly of the Siamese Fighting Fish (Betta splendens) in a University Master’s Course

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401205 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2179-2183 ◽

Cited By ~ 1

Author(s):

Stefan Prost ◽

Malte Petersen ◽

Martin Grethlein ◽

Sarah Joy Hahn ◽

Nina Kuschik-Maczollek ◽

...

Keyword(s):

Genome Assembly ◽

High Throughput Sequencing ◽

Siamese Fighting Fish ◽

Betta Splendens ◽

High Quality ◽

Sequencing Platform ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Chromosome Level

Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behavior. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ∼35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 96.1% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master’s course. The use of ∼35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

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Improving the chromosome-level genome assembly of the Siamese fighting fish (Betta splendens) in a university Master’s course

10.1101/2020.03.06.981332 ◽

2020 ◽

Author(s):

Stefan Prost ◽

Malte Petersen ◽

Martin Grethlein ◽

Sarah Joy Hahn ◽

Nina Kuschik-Maczollek ◽

...

Keyword(s):

Genome Assembly ◽

High Throughput Sequencing ◽

Siamese Fighting Fish ◽

Betta Splendens ◽

High Quality ◽

Sequencing Platform ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Chromosome Level

AbstractBackgroundEver decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university Master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behaviour. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published HiC data.FindingsThe use of nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using previously published HiC data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 95.8% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly.ConclusionWe present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university Master’s course. The use of ~35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

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NGSpeciesID: DNA barcode and amplicon consensus generation from long-read sequencing data

10.22541/au.160262406.62842291/v2 ◽

2020 ◽

Author(s):

Kristoffer Sahlin ◽

Marisa Lim ◽

Stefan Prost

Keyword(s):

High Throughput Sequencing ◽

Dna Barcode ◽

Amplicon Sequencing ◽

Error Rates ◽

Sequencing Data ◽

Sequencing Platform ◽

Consensus Sequences ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read

Third generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long read sequences. This is not only advantageous for genome sequencing projects, but also for amplicon-based high-throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high quality consensus sequences. Here we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long-read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines

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

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqab034 ◽

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.

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Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions

Genes ◽

10.3390/genes10110858 ◽

2019 ◽

Vol 10 (11) ◽

pp. 858 ◽

Cited By ~ 18

Author(s):

Krehenwinkel ◽

Pomerantz ◽

Prost

Keyword(s):

Dna Sequencing ◽

Biodiversity Loss ◽

Taxonomic Composition ◽

Great Promise ◽

Sequencing Platform ◽

Biological Communities ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Sequencing Studies ◽

High Throughput Dna Sequencing

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development.

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Swan: a library for the analysis and visualization of long-read transcriptomes

Bioinformatics ◽

10.1093/bioinformatics/btaa836 ◽

2020 ◽

Author(s):

Fairlie Reese ◽

Ali Mortazavi

Keyword(s):

Rna Sequencing ◽

Cell Lines ◽

Intron Retention ◽

Exon Skipping ◽

Differentially Expressed ◽

Transcript Isoforms ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read

Abstract Motivation Long-read RNA-sequencing technologies such as PacBio and Oxford Nanopore have discovered an explosion of new transcript isoforms that are difficult to visually analyze using currently available tools. We introduce the Swan Python library, which is designed to analyze and visualize transcript models. Results Swan finds 4909 differentially expressed transcripts between cell lines HepG2 and HFFc6, including 279 that are differentially expressed even though the parent gene is not. Additionally, Swan discovers 285 reproducible exon skipping and 47 intron retention events not recorded in the GENCODE v29 annotation. Availability and implementation The Swan library for Python 3 is available on PyPi at https://pypi.org/project/swan-vis/ and on GitHub at https://github.com/mortazavilab/swan_vis.

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Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

Frontiers in Genetics ◽

10.3389/fgene.2021.761791 ◽

2021 ◽

Vol 12 ◽

Author(s):

Davide Bolognini ◽

Alberto Magi

Keyword(s):

Variant Calling ◽

Research Report ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Factors Affecting ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Sequencing Studies ◽

Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

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Updated Genome Sequence for the Probiotic Bacterium Bifidobacterium animalis subsp. lactis BB-12

Microbiology Resource Announcements ◽

10.1128/mra.00078-21 ◽

2021 ◽

Vol 10 (27) ◽

Author(s):

Kristian Jensen ◽

Kosai Al-Nakeeb ◽

Anna Koza ◽

Ahmad A. Zeidan

Keyword(s):

Genome Sequence ◽

Probiotic Bacterium ◽

Content Type ◽

Short Read Sequencing ◽

Bifidobacterium Animalis ◽

Oxford Nanopore ◽

Hybrid Genome ◽

Long Read ◽

Sequencing Platforms ◽

Oxford Nanopore Technologies

The genome of Bifidobacterium animalis subsp. lactis BB-12 was sequenced using Oxford Nanopore Technologies long-read and Illumina short-read sequencing platforms. A hybrid genome assembly approach was used to construct an updated complete genome sequence for BB-12 containing 1,944,152 bp, with a G+C content of 60.5% and 1,615 genes.

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Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality

F1000Research ◽

10.12688/f1000research.11022.3 ◽

2017 ◽

Vol 6 ◽

pp. 227 ◽

Cited By ~ 1

Author(s):

Scott Gigante

Keyword(s):

Data Storage ◽

Data Generation ◽

Biologically Relevant ◽

Sequencing Technologies ◽

Long Term Storage ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies ◽

Term Storage

Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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Long read sequencing reveals Poxvirus evolution through rapid homogenization of gene arrays

10.1101/245373 ◽

2018 ◽

Author(s):

Thomas A. Sasani ◽

Kelsey R. Cone ◽

Aaron R. Quinlan ◽

Nels C. Elde

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Single Nucleotide Variants ◽

Sequencing Platform ◽

Sequencing Technologies ◽

Gene Copy Number Variation ◽

Large Gene ◽

Long Read ◽

Number Variation

AbstractLarge DNA viruses rapidly evolve to defeat host defenses. Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same locus, yet how these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to immune blocks is unknown. We performed experimental evolution with a vaccinia virus population harboring a SNV in a gene actively undergoing copy number amplification. Comparisons of virus genomes using the Oxford Nanopore Technologies sequencing platform allowed us to phase SNVs within large gene copy arrays for the first time, and uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. Our work reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses, and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

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