scholarly journals Taxonomic resolution of the ribosomal RNA operon in bacteria: Implications for its use with long read sequencing

2019 ◽  
Author(s):  
Leonardo de Oliveira Martins ◽  
Andrew J. Page ◽  
Ian G. Charles
Keyword(s):  
Ribosomal Rna ◽  
Sequence Variation ◽  
Full Length ◽  
Taxonomic Resolution ◽  
Bacterial Cells ◽  
Genus Level ◽  
Sequencing Technologies ◽  
Ribosomal Operon ◽  
Long Read ◽  
Multiple Copies

AbstractLong-read sequencing technologies enable capture of the full-length of ribosomal RNA operons in a single read. Bacterial cells usually have multiple copies of this ribosomal operon; sequence variation within a species of bacterium can exceed variation between species. For uncultured organisms this may affect the overall taxonomic resolution, to genus level, of the full-length ribosomal operon.

scholarly journals Taxonomic resolution of the ribosomal RNA operon in bacteria: implications for its use with long-read sequencing

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Leonardo de Oliveira Martins ◽  
Andrew J Page ◽  
Alison E Mather ◽  
Ian G Charles
Keyword(s):  
Phylogenetic Signal ◽  
Taxonomic Diversity ◽  
Taxonomic Resolution ◽  
Bacterial Cells ◽  
Species Classification ◽  
Sequencing Technologies ◽  
Ribosomal Operon ◽  
Long Read ◽  
Multiple Copies ◽  
Ribosomal Operons

Abstract DNA barcoding through the use of amplified regions of the ribosomal operon, such as the 16S gene, is a routine method to gain an overview of the microbial taxonomic diversity within a sample without the need to isolate and culture the microbes present. However, bacterial cells usually have multiple copies of this ribosomal operon, and choosing the ‘wrong’ copy could provide a misleading species classification. While this presents less of a problem for well-characterized organisms with large sequence databases to interrogate, it is a significant challenge for lesser known organisms with unknown copy number and diversity. Using the entire length of the ribosomal operon, which encompasses the 16S, 23S, 5S and internal transcribed spacer regions, should provide greater taxonomic resolution but has not been well explored. Here, we use publicly available reference genomes and explore the theoretical boundaries when using concatenated genes and the full-length ribosomal operons, which has been made possible by the development and uptake of long-read sequencing technologies. We quantify the issues of both copy choice and operon length in a phylogenetic context to demonstrate that longer regions improve the phylogenetic signal while maintaining taxonomic accuracy.

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 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.

scholarly journals SVants – A long-read based method for structural variation detection in bacterial genomes

2019 ◽  
Author(s):  
BM Hanson ◽  
JS Johnson ◽  
SR Leopold ◽  
E Sodergren ◽  
GM Weinstock
Keyword(s):  
Structural Variation ◽  
Tandem Repeats ◽  
Bacterial Genome ◽  
Genetic Material ◽  
Bacterial Cells ◽  
Sequencing Data ◽  
E Coli ◽  
Sequencing Technologies ◽  
Long Read ◽  
New Locations

AbstractMotivationMobile genetic elements (MGEs) are genetic material that can transfer between bacterial cells and move to new locations within a single bacterial genome. These elements range from several hundred to tens of thousands of bases, and are often bordered by repeat regions, which makes resolving these elements difficult with short-read sequencing data. The development and availability of long-read sequencing technologies has opened up new opportunities in the study of structural variation but there is a lack of bioinformatics tools designed to take advantage of these longer reads.ResultsWe present an assembly-free method for identifying the location of these MGEs when compared to any reference genome (including draft genomes). Using an artificially constructed Escherichia coli genome containing single and tandem-repeats of a Tn9 transposon, we demonstrate the ability of SVants to accurately identify multiple insertion sites as well as count the number of repeats of this MGE. Additionally, we show that SVants accurately identifies the transposon of interest, Tn9, but does not erroneously identify existing IS1 regions present within the chromosome of the E. coli artificial reference.Availability and ImplementationSVants is available as open-source software at https://github.com/EpiBlake/SVants

Identification of Full-length Circular Nucleic Acids using Long-read Sequencing Technologies

The Analyst ◽  
2021 ◽  
Author(s):  
Wenxiang Lu ◽  
Kequan Yu ◽  
Xiaohan Li ◽  
Qinyu Ge ◽  
Geyu Liang ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Genomic Dna ◽  
Full Length ◽  
Sequencing Technologies ◽  
Circular Configuration ◽  
Long Read

Unlike the traditional perception in genomic DNA or linear RNA, circular nucleic acids are a class of functional biomolecules with a circular configuration and are often observed in nature. These...

scholarly journals Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

2021 ◽  
Author(s):  
Camilla Ugolini ◽  
Logan Mulroney ◽  
Adrien Leger ◽  
Matteo Castelli ◽  
Elena Criscuolo ◽  
...  
Keyword(s):  
Viral Genome ◽  
Full Length ◽  
Accessory Proteins ◽  
Genomic Rnas ◽  
Robust Estimates ◽  
Sequencing Technologies ◽  
Junction Site ◽  
Long Read ◽  
A New Technique ◽  
Viral Isolates

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

scholarly journals Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

2019 ◽  
Author(s):  
Mahwash Jamy ◽  
Rachel Foster ◽  
Pierre Barbera ◽  
Lucas Czech ◽  
Alexey Kozlov ◽  
...  
Keyword(s):  
Phylogenetic Signal ◽  
Large Subunit ◽  
Environmental Dna ◽  
Taxonomic Resolution ◽  
Soil Dna ◽  
Environmental Diversity ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Phylogenetic Resolution

AbstractHigh-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order to assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based and phylogenetic placement-based methods using shorter reads. The taxonomically-annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing to accurately derive the evolutionary origin of environmental diversity. A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

scholarly journals FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yanping Long ◽  
Zhijian Liu ◽  
Jinbu Jia ◽  
Weipeng Mo ◽  
Liang Fang ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Walls ◽  
Large Scale ◽  
Full Length ◽  
Cell Level ◽  
Root Cells ◽  
Rna Profiling ◽  
Different Types ◽  
Long Read ◽  
Single Nucleus

AbstractThe broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

scholarly journals Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

2021 ◽  
Author(s):  
Valentin Waschulin ◽  
Chiara Borsetto ◽  
Robert James ◽  
Kevin K. Newsham ◽  
Stefano Donadio ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Full Length ◽  
Metagenomic Sequencing ◽  
Short Read ◽  
Short Read Sequencing ◽  
Rich Diversity ◽  
Long Read ◽  
The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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