scholarly journals Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

2015 ◽  
Vol 5 ◽  
Author(s):  
Alexander W. Eastman ◽  
Ze-Chun Yuan
Keyword(s):  
De Novo ◽  
Bacterial Genome ◽  
Genome Finishing ◽  
Development And Validation

scholarly journals riboSeed: leveraging prokaryotic genomic architecture to assemble across ribosomal regions

2017 ◽  
Author(s):  
Nicholas R. Waters ◽  
Florence Abram ◽  
Fiona Brennan ◽  
Ashleigh Holmes ◽  
Leighton Pritchard
Keyword(s):  
De Novo ◽  
Bacterial Genome ◽  
Genomic Context ◽  
Inherent Difficulty ◽  
Short Reads ◽  
Genomic Architecture ◽  
A Genome ◽  
Ribosomal Operons ◽  
Flanking Regions ◽  
Bacterial Genome Sequencing

The vast majority of bacterial genome sequencing has been performed using Illumina short reads. Because of the inherent difficulty of resolving repeated regions with short reads alone, only ≈10% of sequencing projects have resulted in a closed genome. The most common repeated regions are those coding for ribosomal operons (rDNAs), which occur in a bacterial genome between 1 and 15 times, and are typically used as sequence markers to classify and identify bacteria. Here, we exploit conservation in the genomic context in which rDNAs occur across taxa to improve assembly of these regions relative to de novo sequencing by using the conserved nature of rDNAs across taxa and the uniqueness of their flanking regions within a genome. We describe a method to construct targeted pseudocontigs generated by iteratively assembling reads that map to a reference genome’s rDNAs. These pseudocontigs are then used to more accurately assemble the newly-sequenced chromosome. We show that this method, implemented as riboSeed, correctly bridges across adjacent contigs in bacterial genome assembly and, when used in conjunction with other genome polishing tools, can assist in closure of a genome.

scholarly journals Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules

2021 ◽  
Author(s):  
Seyed Mohammad Siadat ◽  
Monica E Susilo ◽  
Jeffrey A Paten ◽  
Alexandra A Silverman ◽  
Charles A DiMarzio ◽  
...  
Keyword(s):  
Type I Collagen ◽  
De Novo ◽  
Type I ◽  
Growth And Remodeling ◽  
Fibril Structure ◽  
Collagen Molecules ◽  
Vitro Model ◽  
Development And Validation ◽  
Network Morphology

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.

scholarly journals Evaluation of hybrid and non-hybrid methods for de novo assembly of nanopore reads

2015 ◽  
Author(s):  
Ivan Sovic ◽  
Kresimir Krizanovic ◽  
Karolj Skala ◽  
Mile Sikic
Keyword(s):  
De Novo Assembly ◽  
De Novo ◽  
Hybrid Methods ◽  
Bacterial Genome ◽  
Error Rates ◽  
Sequencing Data ◽  
E Coli ◽  
Recent Emergence ◽  
K 12

Recent emergence of nanopore sequencing technology set a challenge for the established assembly methods not optimized for the combination of read lengths and high error rates of nanopore reads. In this work we assessed how existing de novo assembly methods perform on these reads. We benchmarked three non-hybrid (in terms of both error correction and scaffolding) assembly pipelines as well as two hybrid assemblers which use third generation sequencing data to scaffold Illumina assemblies. Tests were performed on several publicly available MinION and Illumina datasets of E. coli K-12, using several sequencing coverages of nanopore data (20x, 30x, 40x and 50x). We attempted to assess the quality of assembly at each of these coverages, to estimate the requirements for closed bacterial genome assembly. Results show that hybrid methods are highly dependent on the quality of NGS data, but much less on the quality and coverage of nanopore data and perform relatively well on lower nanopore coverages. Furthermore, when coverage is above 40x, all non-hybrid methods correctly assemble the E. coli genome, even a non-hybrid method tailored for Pacific Bioscience reads. While it requires higher coverage compared to a method designed particularly for nanopore reads, its running time is significantly lower.

scholarly journals High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
John M. Urban ◽  
Michael S. Foulk ◽  
Jacob E. Bliss ◽  
C. Michelle Coleman ◽  
Nanyan Lu ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Single Molecule ◽  
De Novo ◽  
Bacterial Genome ◽  
Draft Genome ◽  
Dna Amplification ◽  
Chromosome Elimination ◽  
Paternal Chromosome ◽  
De Novo Genome Assembly ◽  
Long Read

Abstract Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

scholarly journals A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer

2014 ◽  
Author(s):  
Josh Quick ◽  
Aaron Quinlan ◽  
Nicholas Loman
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Sequence Data ◽  
Bacterial Genome ◽  
Model Organism ◽  
Variant Calling ◽  
Laptop Computer ◽  
Early Access ◽  
Dna Strands ◽  
K 12

Background: The MinION™ is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. By measuring the change in current produced when DNA strands translocate through and interact with a charged protein nanopore the device is able to deduce the underlying nucleotide sequence. Findings: We present a read dataset from whole-genome shotgun sequencing of the model organism Escherichia coli K-12 substr. MG1655 generated on a MinION™ device during the early-access MinION Access Program (MAP). Sequencing runs of the MinION™ are presented, one generated using R7 chemistry (released in July 2014) and one using R7.3 (released in September 2014). Conclusions: Base-called sequence data are provided to demonstrate the nature of data produced by the MinION™ platform and to encourage the development of customised methods for alignment, consensus and variant calling, de novo assembly and scaffolding. FAST5 files containing event data within the HDF5 container format are provided to assist with the development of improved base-calling methods. Datasets are provided through the GigaDB database at http://gigadb.org/dataset/100102

scholarly journals De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer

2008 ◽  
Vol 18 (5) ◽  
pp. 802-809 ◽  
Author(s):  
D. Hernandez ◽  
P. Francois ◽  
L. Farinelli ◽  
M. Osteras ◽  
J. Schrenzel
Keyword(s):  
Genome Sequencing ◽  
De Novo ◽  
Bacterial Genome ◽  
Desktop Computer ◽  
Short Reads ◽  
Bacterial Genome Sequencing

Development and validation of EST-SSR markers of Magnolia wufengensis using de novo transcriptome sequencing

Trees ◽  
2019 ◽  
Vol 33 (4) ◽  
pp. 1213-1223 ◽  
Author(s):  
Li Wang ◽  
Xiaoqiang Gong ◽  
Lin Jin ◽  
Haiying Li ◽  
Junqi Lu ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Transcriptome Sequencing ◽  
De Novo ◽  
De Novo Transcriptome ◽  
De Novo Transcriptome Sequencing ◽  
Development And Validation ◽  
Magnolia Wufengensis

TORMES: an automated pipeline for whole bacterial genome analysis

2019 ◽  
Vol 35 (21) ◽  
pp. 4207-4212 ◽  
Author(s):  
Narciso M Quijada ◽  
David Rodríguez-Lázaro ◽  
Jose María Eiros ◽  
Marta Hernández
Keyword(s):  
Genome Sequencing ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Sequence Data ◽  
Bacterial Genome ◽  
Draft Genome ◽  
Supplementary Information ◽  
Computing Skills ◽  
Quality Filtering ◽  
Sequence Quality

Abstract Motivation The progress of High Throughput Sequencing (HTS) technologies and the reduction in the sequencing costs are such that Whole Genome Sequencing (WGS) could replace many traditional laboratory assays and procedures. Exploiting the volume of data produced by HTS platforms requires substantial computing skills and this is the main bottleneck in the implementation of WGS as a routine laboratory technique. The way in which the vast amount of results are presented to researchers and clinicians with no specialist knowledge of genome sequencing is also a significant issue. Results Here we present TORMES, a user-friendly pipeline for WGS analysis of bacteria from any origin generated by HTS on Illumina platforms. TORMES is designed for non-bioinformatician users, and automates the steps required for WGS analysis directly from the raw sequence data: sequence quality filtering, de novo assembly, draft genome ordering against a reference, genome annotation, multi-locus sequence typing (MLST), searching for antibiotic resistance and virulence genes, and pangenome comparisons. Once the analysis is finished, TORMES generates and interactive web-like report that can be opened in any web browser and shared and revised by researchers in a simple manner. TORMES can be run by using very simple commands and represent a quick an easy way to perform WGS analysis. Availability and implementation TORMES is free available at https://github.com/nmquijada/tormes. Supplementary information Supplementary data are available at Bioinformatics online.

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