scholarly journals Generating closed bacterial genomes from long-read nanopore sequencing of microbiomes

2018 ◽  
Author(s):  
Eli L. Moss ◽  
Ami S. Bhatt
Keyword(s):  
Genome Structure ◽  
Nanopore Sequencing ◽  
Bacterial Genomes ◽  
Human Gut ◽  
Healthy Human ◽  
Short Read ◽  
Long Read ◽  
Efficient Recovery

AbstractWe present the first method for efficient recovery of complete, closed genomes directly from microbiomes using nanopore long-read sequencing and assembly. We apply our approach to three healthy human gut communities and compare results to short read and read cloud approaches. We obtain nine finished genomes including the first reported closed genome of Prevotella copri, an organism with highly repetitive genome structure prevalent in non-western human gut microbiomes.

scholarly journals Complete, closed bacterial genomes from microbiomes using nanopore sequencing

2020 ◽  
Vol 38 (6) ◽  
pp. 701-707 ◽  
Author(s):  
Eli L. Moss ◽  
Dylan G. Maghini ◽  
Ami S. Bhatt
Keyword(s):  
Bacterial Species ◽  
Genome Structure ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Bacterial Genomes ◽  
Short Read ◽  
Repeat Elements ◽  
Long Read ◽  
Metagenomics Data ◽  
Read Error Correction

AbstractMicrobial genomes can be assembled from short-read sequencing data, but the assembly contiguity of these metagenome-assembled genomes is constrained by repeat elements. Correct assignment of genomic positions of repeats is crucial for understanding the effect of genome structure on genome function. We applied nanopore sequencing and our workflow, named Lathe, which incorporates long-read assembly and short-read error correction, to assemble closed bacterial genomes from complex microbiomes. We validated our approach with a synthetic mixture of 12 bacterial species. Seven genomes were completely assembled into single contigs and three genomes were assembled into four or fewer contigs. Next, we used our methods to analyze metagenomics data from 13 human stool samples. We assembled 20 circular genomes, including genomes of Prevotella copri and a candidate Cibiobacter sp. Despite the decreased nucleotide accuracy compared with alternative sequencing and assembly approaches, our methods improved assembly contiguity, allowing for investigation of the role of repeat elements in microbial function and adaptation.

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 Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing

2018 ◽  
Author(s):  
Natalie Ring ◽  
Jonathan Abrahams ◽  
Miten Jain ◽  
Hugh Olsen ◽  
Andrew Preston ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Flow Cell ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Genome Sequences ◽  
Short Read ◽  
Link Type ◽  
Analysis Tools ◽  
Long Read ◽  
Assembly Pipeline

ABSTRACTThe genome of Bordetella pertussis is complex, with high GC content and many repeats, each longer than 1,000 bp. Short-read DNA sequencing is unable to resolve the structure of the genome; however, long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussis strains in a single sequencing run. We then trialled combinations of the many nanopore-user-community-built long-read analysis tools to establish the current optimal assembly pipeline for B. pertussis genome sequences. Our best long-read-only assemblies were produced by Canu read correction followed by assembly with Flye and polishing with Nanopolish, whilst the best hybrids (using nanopore and Illumina reads together) were produced by Canu correction followed by Unicycler. This pipeline produced closed genome sequences for four strains, revealing inter-strain genomic rearrangement. However, read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (over 100 kbp), which was not resolved by our pipeline. We have therefore demonstrated the ability to resolve the structure of several B. pertussis strains per single barcoded nanopore flow cell, but the genomes with highest complexity (e.g. very large duplicated regions) remain only partially resolved using the standard library preparation and will require an alternative library preparation method. For full strain characterisation, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.DATA SUMMARYFinal sequence read files (fastq) for all 5 strains have been deposited in the SRA, BioProject PRJNA478201, accession numbers SAMN09500966, SAMN09500967, SAMN09500968, SAMN09500969, SAMN09500970A full list of accession numbers for Illumina sequence reads is available in Table S1Assembly tests, basecalled read sets and reference materials are available from figshare: https://figshare.com/projects/Resolving_the_complex_Bordetella_pertussis_genome_using_barcoded_nanopore_sequencing/31313Genome sequences for B. pertussis strains UK36, UK38, UK39, UK48 and UK76 have been deposited in GenBank; accession numbers: CP031289, CP031112, CP031113, QRAX00000000, CP031114Source code and full commands used are available from Github: https://github.com/nataliering/Resolving-the-complex-Bordetella-pertussis-genome-using-barcoded-nanopore-sequencingIMPACT STATEMENTOver the past two decades, whole genome sequencing has allowed us to understand microbial pathogenicity and evolution on an unprecedented level. However, repetitive regions, like those found throughout the B. pertussis genome, have confounded our ability to resolve complex genomes using short-read sequencing technologies alone. To produce closed B. pertussis genome sequences it is necessary to use a sequencing technology which can generate reads longer than these problematic genomic regions. Using barcoded nanopore sequencing, we show that multiple B. pertussis genomes can be resolved per flow cell. Use of our assembly pipeline to resolve further B. pertussis genomes will advance understanding of how genome-level differences affect the phenotypes of strains which appear monomorphic at nucleotide-level.This work expands the recently emergent theme that even the most complex genomes can be resolved with sufficiently long sequencing reads. Additionally, we utilise a more widely accessible alternative sequencing platform to the Pacific Biosciences platform already used by large research centres such as the CDC. Our optimisation process, moreover, shows that the analysis tools favoured by the sequencing community do not necessarily produce the most accurate assemblies for all organisms; pipeline optimisation may therefore be beneficial in studies of unusually complex genomes.

scholarly journals Long-read sequencing reveals atypical mitochondrial genome structure in a New Zealand marine isopod

2022 ◽  
Vol 9 (1) ◽  
Author(s):  
William S. Pearman ◽  
Sarah J. Wells ◽  
James Dale ◽  
Olin K. Silander ◽  
Nikki E. Freed
Keyword(s):  
New Zealand ◽  
Mitochondrial Genome ◽  
Dna Sequencing ◽  
Recent Work ◽  
Genome Structure ◽  
Mitochondrial Genomes ◽  
Short Read ◽  
Mitochondrial Structure ◽  
Evolutionary Origins ◽  
Long Read

Most animal mitochondrial genomes are small, circular and structurally conserved. However, recent work indicates that diverse taxa possess unusual mitochondrial genomes. In Isopoda , species in multiple lineages have atypical and rearranged mitochondrial genomes. However, more species of this speciose taxon need to be evaluated to understand the evolutionary origins of atypical mitochondrial genomes in this group. In this study, we report the presence of an atypical mitochondrial structure in the New Zealand endemic marine isopod, Isocladus armatus. Data from long- and short-read DNA sequencing suggest that I. armatus has two mitochondrial chromosomes. The first chromosome consists of two mitochondrial genomes that have been inverted and fused together in a circular form, and the second chromosome consists of a single mitochondrial genome in a linearized form. This atypical mitochondrial structure has been detected in other isopod lineages, and our data from an additional divergent isopod lineage (Sphaeromatidae) lends support to the hypothesis that atypical structure evolved early in the evolution of Isopoda . Additionally, we find that an asymmetrical site previously observed across many species within Isopoda is absent in I. armatus , but confirm the presence of two asymmetrical sites recently reported in two other isopod species.

scholarly journals Nanopype: a modular and scalable nanopore data processing pipeline

2019 ◽  
Vol 35 (22) ◽  
pp. 4770-4772
Author(s):  
Pay Giesselmann ◽  
Sara Hetzel ◽  
Franz-Josef Müller ◽  
Alexander Meissner ◽  
Helene Kretzmer
Keyword(s):  
Data Processing ◽  
Supplementary Information ◽  
Nanopore Sequencing ◽  
Third Generation ◽  
Supplementary Data ◽  
Seamless Integration ◽  
Short Read ◽  
Processing Pipeline ◽  
Bioinformatics Software ◽  
Long Read

Abstract Summary Long-read third-generation nanopore sequencing enables researchers to now address a range of questions that are difficult to tackle with short read approaches. The rapidly expanding user base and continuously increasing throughput have sparked the development of a growing number of specialized analysis tools. However, streamlined processing of nanopore datasets using reproducible and transparent workflows is still lacking. Here we present Nanopype, a nanopore data processing pipeline that integrates a diverse set of established bioinformatics software while maintaining consistent and standardized output formats. Seamless integration into compute cluster environments makes the framework suitable for high-throughput applications. As a result, Nanopype facilitates comparability of nanopore data analysis workflows and thereby should enhance the reproducibility of biological insights. Availability and implementation https://github.com/giesselmann/nanopype, https://nanopype.readthedocs.io. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Long read sequencing reveals atypical mitochondrial genome structure in a New Zealand marine isopod

2021 ◽  
Author(s):  
William S Pearman ◽  
Sarah J Wells ◽  
James Dale ◽  
Olin K Silander ◽  
Nikki E Freed
Keyword(s):  
New Zealand ◽  
Mitochondrial Genome ◽  
Dna Sequencing ◽  
Recent Work ◽  
Genome Structure ◽  
Mitochondrial Genomes ◽  
Short Read ◽  
Mitochondrial Structure ◽  
Evolutionary Origins ◽  
Long Read

Most animal mitochondrial genomes are small, circular, and structurally conserved. However, recent work indicates that diverse taxa possess unusual mitochondrial genomes. In Isopoda, species in multiple lineages have atypical and rearranged mitochondrial genomes. However, more species of this speciose taxon need to be evaluated to understand the evolutionary origins of atypical mitochondrial genomes in this group. In this study, we report the presence of an atypical mitochondrial structure in the New Zealand endemic marine isopod, Isocladus armatus. Data from long and short read DNA sequencing, suggests that I. armatus has two mitochondrial chromosomes. The first chromosome consists of two mitochondrial genomes that have been inverted and fused together in a circular form, and the second chromosome consists of a single mitochondrial genome in a linearized form. This atypical mitochondrial structure has been detected in other isopod lineages, and our data from an additional divergent isopod lineage (Sphaeromatidae) lends support to the hypothesis that atypical structure evolved early in the evolution of Isopoda. Additionally, we find that a heteroplasmic site previously observed across many species within Isopoda is absent in I. armatus, but confirm the presence of two heteroplasmic sites recently reported in two other isopod species.

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 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 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 metaFlye: scalable long-read metagenome assembly using repeat graphs

2019 ◽  
Author(s):  
Mikhail Kolmogorov ◽  
Mikhail Rayko ◽  
Jeffrey Yuan ◽  
Evgeny Polevikov ◽  
Pavel Pevzner
Keyword(s):  
Dark Matter ◽  
State Of The Art ◽  
Full Length ◽  
Bacterial Genomes ◽  
Short Read ◽  
Sequencing Technologies ◽  
16S Rna ◽  
Long Read ◽  
Metagenome Assembly ◽  
Rna Genes

AbstractLong-read sequencing technologies substantially improved assemblies of many isolate bacterial genomes as compared to fragmented assemblies produced with short-read technologies. However, assembling complex metagenomic datasets remains a challenge even for the state-of-the-art long-read assemblers. To address this gap, we present the metaFlye assembler and demonstrate that it generates highly contiguous and accurate metagenome assemblies. In contrast to short-read metagenomics assemblers that typically fail to reconstruct full-length 16S RNA genes, metaFlye captures many 16S RNA genes within long contigs, thus providing new opportunities for analyzing the microbial “dark matter of life”. We also demonstrate that long-read metagenome assemblers significantly improve full-length plasmid and virus reconstruction as compared to short-read assemblers and reveal many novel plasmids and viruses.

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