scholarly journals Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

2021 ◽  
Author(s):  
Yelena Chernyavskaya ◽  
Xiaofei Zhang ◽  
Jinze Liu ◽  
Jessica S. Blackburn
Keyword(s):  
Low Complexity ◽  
Zebrafish Genome ◽  
Nanopore Sequencing ◽  
Sequencing Technology ◽  
Short Read ◽  
Short Read Sequencing ◽  
Genomic Landscape ◽  
Long Reads ◽  
Long Read ◽  
Sequencing Platforms

Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.

scholarly journals Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

scholarly journals Closed Genome Sequence of Salmonella enterica Serovar Richmond Strain CFSAN000191, Obtained with Nanopore Sequencing

2018 ◽  
Vol 7 (23) ◽  
Author(s):  
Narjol González-Escalona ◽  
Kuan Yao ◽  
Maria Hoffmann
Keyword(s):  
Genome Sequence ◽  
Salmonella Enterica ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Content Type ◽  
Short Read Sequencing ◽  
Long Read

Here we report the genome sequence of Salmonella enterica serovar Richmond strain CFSAN000191, isolated from tilapia from Thailand in 2005. The genome was determined by a combination of long-read and short-read sequencing.

scholarly journals CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0241253
Author(s):  
Amelia D. Wallace ◽  
Thomas A. Sasani ◽  
Jordan Swanier ◽  
Brooke L. Gates ◽  
Jeff Greenland ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Tandem Repeats ◽  
Short Read ◽  
Background Elimination ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Repeat Expansions ◽  
Sequencing Platforms ◽  
Als Patients

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39–416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

scholarly journals Highly accurate barcode and UMI error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Short Read Sequencing ◽  
Single Cell Sequencing ◽  
Base Calling ◽  
Novel Approach ◽  
Long Read ◽  
First Time ◽  
Insight Into

AbstractDroplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

scholarly journals Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes

2019 ◽  
Author(s):  
Nicola De Maio ◽  
Liam P. Shaw ◽  
Alasdair Hubbard ◽  
Sophie George ◽  
Nick Sanderson ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Hybrid Assembly ◽  
Bacterial Genomes ◽  
Short Read ◽  
Short Reads ◽  
Genome Reconstruction ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sequencing Platforms

ABSTRACTIllumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods impact on assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or from SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the Enterobacteriaceae family, as these frequently have highly plastic, repetitive genetic structures and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies. Both strategies facilitate high-quality genome reconstruction. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.IMPACT STATEMENTIllumina short-read sequencing is frequently used for tasks in bacterial genomics, such as assessing which species are present within samples, checking if specific genes of interest are present within individual isolates, and reconstructing the evolutionary relationships between strains. However, while short-read sequencing can reveal significant detail about the genomic content of bacterial isolates, it is often insufficient for assessing genomic structure: how different genes are arranged within genomes, and particularly which genes are on plasmids – potentially highly mobile components of the genome frequently carrying antimicrobial resistance elements. This is because Illumina short reads are typically too short to span repetitive structures in the genome, making it impossible to accurately reconstruct these repetitive regions. One solution is to complement Illumina short reads with long reads generated with SMRT Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) sequencing platforms. Using this approach, called ‘hybrid assembly’, we show that we can automatically fully reconstruct complex bacterial genomes of Enterobacteriaceae isolates in the majority of cases (best-performing method: 17/20 isolates). In particular, by comparing different methods we find that using the assembler Unicycler with Illumina and ONT reads represents a low-cost, high-quality approach for reconstructing bacterial genomes using publicly available software.DATA SUMMARYRaw sequencing data and assemblies have been deposited in NCBI under BioProject Accession PRJNA422511 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA422511). We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

scholarly journals Realizing the potential of full-length transcriptome sequencing

2019 ◽  
Vol 374 (1786) ◽  
pp. 20190097 ◽  
Author(s):  
Ashley Byrne ◽  
Charles Cole ◽  
Roger Volden ◽  
Christopher Vollmers
Keyword(s):  
Single Cell ◽  
Transcriptome Analysis ◽  
Transcriptome Sequencing ◽  
Model Organisms ◽  
Sequencing Technology ◽  
Short Read ◽  
Short Read Sequencing ◽  
Unicellular Eukaryotes ◽  
Long Read ◽  
Future Work

Long-read sequencing holds great potential for transcriptome analysis because it offers researchers an affordable method to annotate the transcriptomes of non-model organisms. This, in turn, will greatly benefit future work on less-researched organisms like unicellular eukaryotes that cannot rely on large consortia to generate these transcriptome annotations. However, to realize this potential, several remaining molecular and computational challenges will have to be overcome. In this review, we have outlined the limitations of short-read sequencing technology and how long-read sequencing technology overcomes these limitations. We have also highlighted the unique challenges still present for long-read sequencing technology and provided some suggestions on how to overcome these challenges going forward. This article is part of a discussion meeting issue ‘Single cell ecology’.

scholarly journals Nanopore sequencing enables high-resolution analysis of resistance determinants and mobile elements in the human gut microbiome

2018 ◽  
Author(s):  
Denis Bertrand ◽  
Jim Shaw ◽  
Manesh Kalathiappan ◽  
Amanda Hui Qi Ng ◽  
Senthil Muthiah ◽  
...  
Keyword(s):  
Complex Dynamics ◽  
Human Microbiome ◽  
Abundant Species ◽  
Error Rates ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Long Reads ◽  
Long Read

AbstractThe analysis of information rich whole-metagenome datasets acquired from complex microbial communities is often restricted by the fragmented nature of assembly from short-read sequencing. The availability of long-reads from third-generation sequencing technologies (e.g. PacBio or Oxford Nanopore) can help improve assembly quality in principle, but high error rates and low throughput have limited their application in metagenomics. In this work, we describe the first hybrid metagenomic assembler which combines the advantages of short and long-read technologies, providing an order of magnitude improvement in contiguity compared to short read assemblies, and high base-pair level accuracy. The proposed approach (OPERA-MS) integrates a novel assembly-based metagenome clustering technique with an exact scaffolding algorithm that can efficiently assemble repeat rich sequences. Based on evaluations with defined in vitro communities and virtual gut microbiomes, we show that it is possible to assemble near complete genomes from metagenomes with as little as 9× long read coverage, thus enabling high quality assembly of lowly abundant species (<1%). Furthermore, OPERA-MS’s fine-grained clustering is able to deconvolute and assemble multiple genomes of the same species in a single sample, allowing us to study the complex dynamics of the human microbiome at the sub-species level. Applying nanopore sequencing to gut metagenomes of patients undergoing antibiotic treatment, we show that long reads can be obtained from stool samples in clinical studies to produce more meaningful metagenomic assemblies (up to 200× improvement over short-read assemblies), including the closed assembly of >80 putative plasmid/phage sequences and a 263kbp jumbo phage. Our results highlight that high-quality hybrid assemblies provide an unprecedented view of the gut resistome in these patients, including strain dynamics and identification of novel plasmid sequences.

scholarly journals HIGH RESOLUTION ANNOTATION OF ZEBRAFISH TRANSCRIPTOME USING LONG-READ SEQUENCING

2017 ◽  
Author(s):  
German Nudelman ◽  
Antonio Frasca ◽  
Brandon Kent ◽  
Kirsten Edepli-Sadler ◽  
Stuart C. Sealfon ◽  
...  
Keyword(s):  
Model Organism ◽  
Sequencing Data ◽  
High Confidence ◽  
Short Read ◽  
Short Read Sequencing ◽  
Functional Conservation ◽  
Transcript Quantification ◽  
Before And After ◽  
Long Read ◽  
Sequencing Platforms

ABSTRACTWith the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2748 high confidence novel transcripts that originated from previously unannotated loci and 1835 high confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and non-coding RNAs, isoform switching occurrences and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8 kb transcript spanning multiple miR-430 elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.

scholarly journals Closed Genome Sequences of Two Clostridium botulinum Strains Obtained by Nanopore Sequencing

2018 ◽  
Vol 7 (9) ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Julie Haendiges ◽  
Jesse D. Miller ◽  
Shashi K. Sharma
Keyword(s):  
Environmental Sample ◽  
Clostridium Botulinum ◽  
Clinical Sample ◽  
Nanopore Sequencing ◽  
Genome Sequences ◽  
Short Read ◽  
Content Type ◽  
Short Read Sequencing ◽  
Long Read

Here we report the genome sequences of two toxin-producing Clostridium botuli num strains, one environmental sample (83F) and one clinical sample (CDC51232). The genomes were closed by a combination of long-read and short-read sequencing.

scholarly journals High resolution copy number inference in cancer using short-molecule nanopore sequencing

2021 ◽  
Author(s):  
Timour Baslan ◽  
Sam Kovaka ◽  
Fritz J Sedlazeck ◽  
Yanming Zhang ◽  
Robert Wappel ◽  
...  
Keyword(s):  
Copy Number ◽  
Cost Effective ◽  
Chromosome Analysis ◽  
Ease Of Use ◽  
Precision Oncology ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Platforms

Abstract Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs.

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