scholarly journals Contig annotation tool CAT robustly classifies assembled metagenomic contigs and long sequences

2016 ◽  
Author(s):  
Diego D. Cambuy ◽  
Felipe H. Coutinho ◽  
Bas E. Dutilh
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Taxonomic Classification ◽  
Annotation Tool ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
Long Read ◽  
Micro Organisms ◽  
Taxonomic Annotation

AbstractIn modern-day metagenomics, there is an increasing need for robust taxonomic annotation of long DNA sequences from unknown micro-organisms. Long metagenomic sequences may be derived from assembly of short-read metagenomes, or from long-read single molecule sequencing. Here we introduce CAT, a pipeline for robust taxonomic classification of long DNA sequences. We show that CAT correctly classifies contigs at different taxonomic levels, even in simulated metagenomic datasets that are very distantly related from the sequences in the database. CAT is implemented in Python and the required scripts can be freely downloaded from Github.

scholarly journals Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
F. A. Bastiaan von Meijenfeldt ◽  
Ksenia Arkhipova ◽  
Diego D. Cambuy ◽  
Felipe H. Coutinho ◽  
Bas E. Dutilh
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
Taxonomic Classification ◽  
Classification Method ◽  
Reference Database ◽  
Annotation Tool ◽  
Multiple Signals

Abstract Current-day metagenomics analyses increasingly involve de novo taxonomic classification of long DNA sequences and metagenome-assembled genomes. Here, we show that the conventional best-hit approach often leads to classifications that are too specific, especially when the sequences represent novel deep lineages. We present a classification method that integrates multiple signals to classify sequences (Contig Annotation Tool, CAT) and metagenome-assembled genomes (Bin Annotation Tool, BAT). Classifications are automatically made at low taxonomic ranks if closely related organisms are present in the reference database and at higher ranks otherwise. The result is a high classification precision even for sequences from considerably unknown organisms.

scholarly journals Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Yu-Chih Tsai ◽  
Sean Conlan ◽  
Clayton Deming ◽  
Julia A. Segre ◽  
Heidi H. Kong ◽  
...  
Keyword(s):  
Microbial Community ◽  
Human Skin ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
High Quality ◽  
Single Nucleotide ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
Hybrid Approaches ◽  
Long Read

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.

scholarly journals Transcriptome assembly from long-read RNA-seq alignments with StringTie2

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Sam Kovaka ◽  
Aleksey V. Zimin ◽  
Geo M. Pertea ◽  
Roham Razaghi ◽  
Steven L. Salzberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcriptome Assembly ◽  
Rna Seq ◽  
Ability To Work ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
New Methods ◽  
Long Reads ◽  
Long Read

AbstractRNA sequencing using the latest single-molecule sequencing instruments produces reads that are thousands of nucleotides long. The ability to assemble these long reads can greatly improve the sensitivity of long-read analyses. Here we present StringTie2, a reference-guided transcriptome assembler that works with both short and long reads. StringTie2 includes new methods to handle the high error rate of long reads and offers the ability to work with full-length super-reads assembled from short reads, which further improves the quality of short-read assemblies. StringTie2 is more accurate and faster and uses less memory than all comparable short-read and long-read analysis tools.

scholarly journals Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT

2019 ◽  
Author(s):  
F.A. Bastiaan von Meijenfeldt ◽  
Ksenia Arkhipova ◽  
Diego D. Cambuy ◽  
Felipe H. Coutinho ◽  
Bas E. Dutilh
Keyword(s):  
Dna Sequences ◽  
Real World ◽  
Taxonomic Classification ◽  
Reference Database ◽  
Annotation Tool ◽  
High Quality

ABSTRACTCurrent-day metagenomics increasingly requires taxonomic classification of long DNA sequences and metagenome-assembled genomes (MAGs) of unknown microorganisms. We show that the standard best-hit approach often leads to classifications that are too specific. We present tools to classify high-quality metagenomic contigs (Contig Annotation Tool, CAT) and MAGs (Bin Annotation Tool, BAT) and thoroughly benchmark them with simulated metagenomic sequences that are classified against a reference database where related sequences are increasingly removed, thereby simulating increasingly unknown queries. We find that the query sequences are correctly classified at low taxonomic ranks if closely related organisms are present in the reference database, while classifications are made higher in the taxonomy when closely related organisms are absent, thus avoiding spurious classification specificity. In a real-world challenge, we apply BAT to over 900 MAGs from a recent rumen metagenomics study and classified 97% consistently with prior phylogeny-based classifications, but in a fully automated fashion.

scholarly journals Towards a better understanding of the low recall of insertion variants with short-read based variant callers

2020 ◽  
Author(s):  
Wesley Delage ◽  
Julien Thevenon ◽  
Claire Lemaitre
Keyword(s):  
Gold Standard ◽  
Insertion Site ◽  
Structural Variants ◽  
Genomic Context ◽  
Breakpoint Junction ◽  
Short Read ◽  
Depth Analysis ◽  
Long Read ◽  
The Impact

AbstractSince 2009, numerous tools have been developed to detect structural variants (SVs) using short read technologies. Insertions >50 bp are one of the hardest type to discover and are drastically underrepresented in gold standard variant callsets. The advent of long read technologies has completely changed the situation. In 2019, two independent cross technologies studies have published the most complete variant callsets with sequence resolved insertions in human individuals. Among the reported insertions, only 17 to 37% could be discovered with short-read based tools. In this work, we performed an in-depth analysis of these unprecedented insertion callsets in order to investigate the causes of such failures. We have first established a precise classification of insertion variants according to four layers of characterization: the nature and size of the inserted sequence, the genomic context of the insertion site and the breakpoint junction complexity. Because these levels are intertwined, we then used simulations to characterize the impact of each complexity factor on the recall of several SV callers. Simulations showed that the most impacting factor was the insertion type rather than the genomic context, with various difficulties being handled differently among the tested SV callers, and they highlighted the lack of sequence resolution for most insertion calls. Our results explain the low recall by pointing out several difficulty factors among the observed insertion features and provide avenues for improving SV caller algorithms and their [email protected]

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 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 Advancing Genome-Resolved Metagenomics beyond the Shotgun

mSystems ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Rex R. Malmstrom ◽  
Emiley A. Eloe-Fadrosh
Keyword(s):  
Single Molecule ◽  
Cell Sorting ◽  
Stable Isotope Probing ◽  
Natural Environments ◽  
Natural Ecosystems ◽  
Shotgun Metagenomics ◽  
Short Read ◽  
Future Developments ◽  
Long Read ◽  
Shed Light

ABSTRACT Exploration of environmental microbiomes has shed light on the ecological and evolutionary principles at play in natural ecosystems and has been further accelerated through the reconstruction of population genomes to provide genome-centric context. Yet technical challenges with traditional shotgun metagenomics remain for computationally intense short-read assembly, strain heterogeneity within communities, and depth of coverage required for low-abundance microbes. In this Perspective, we highlight three main avenues for promising future developments, including coupling stable isotope probing and genome-resolved metagenomics, applying fluorescence-activated cell sorting approaches to target mini-metagenomes within a larger community, and utilizing single-molecule long-read and synthetic long-read technology to link mobile elements to host microbial cells. These developments on the horizon will undoubtedly advance genome-resolved metagenomic approaches and enable a better understanding of uncultivated microbes in their natural environments.

scholarly journals Haplotype Diversity and Sequence Heterogeneity of Human Telomeres

2020 ◽  
Author(s):  
Kirill Grigorev ◽  
Jonathan Foox ◽  
Daniela Bezdan ◽  
Daniel Butler ◽  
Jared J. Luxton ◽  
...  
Keyword(s):  
Single Molecule ◽  
Disease Risk ◽  
Haplotype Diversity ◽  
Single Molecule Sequencing ◽  
Sequence Heterogeneity ◽  
Repetitive Nature ◽  
Telomere Sequence ◽  
Long Read ◽  
Motif Composition ◽  
Intractable Problem

AbstractTelomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, whose lengths can be correlated with age and disease risk factors. Given their length and repetitive nature, telomeric regions are not easily reconstructed from short read sequencing, making telomere sequence resolution a very costly and generally intractable problem. Recently, long-read sequencing, with read lengths measuring in hundreds of Kbp, has made it possible to routinely read into telomeric regions and inspect their structure. Here, we describe a framework for extracting telomeric reads from single-molecule sequencing experiments, describing their sequence variation and motifs, and for haplotype inference. We find that long telomeric stretches can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize non-canonical motifs (both previously reported as well as novel), and report the first motif composition maps of human telomeric diplotypes on a multi-Kbp scale.

scholarly journals A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 961
Author(s):  
Kevin McKernan ◽  
Liam Kane ◽  
Yvonne Helbert ◽  
Lei Zhang ◽  
Nathan Houde ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Genomic Diversity ◽  
Sequence Coverage ◽  
Single Molecule Sequencing ◽  
Contiguous Gene ◽  
Long Read ◽  
Interesting Variation

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cluster. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cluster. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to a less contiguous gene cluster and may illuminate the previously proposed evolution of psilocybin synthesis.

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