Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

ABSTRACT Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remain a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Here, we discuss the analysis of data from transposon mutagenesis followed by deep sequencing (Tn-seq) to determine gene disruptions differentially represented in a mutant population and Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real-time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications.

Download Full-text

CAMISIM: Simulating metagenomes and microbial communities

10.1101/300970 ◽

2018 ◽

Cited By ~ 4

Author(s):

Adrian Fritz ◽

Peter Hofmann ◽

Stephan Majda ◽

Eik Dahms ◽

Johannes Dröge ◽

...

Keyword(s):

Microbial Communities ◽

De Novo ◽

Real Data ◽

Small Data ◽

Data Sets ◽

Sequencing Data ◽

Taxonomic Profiling ◽

Benchmark Data ◽

Sequencing Technologies ◽

Wide Range

Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required. Here, we describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series and differential abundance studies, includes real and simulated strain-level diversity, and generates second and third generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT and metaSPAdes, on several thousand small data sets generated with CAMISIM. CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with truth standards for method evaluation. All data sets and the software are freely available at: https://github.com/CAMI-challenge/CAMISIM

Download Full-text

Mapping and phasing of structural variation in patient genomes using nanopore sequencing

10.1101/129379 ◽

2017 ◽

Cited By ~ 4

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.

Download Full-text

Long-Read, Single Molecule, Real-Time (SMRT) DNA Sequencing for Metagenomic Applications

Metagenomics for Microbiology ◽

10.1016/b978-0-12-410472-3.00002-6 ◽

2015 ◽

pp. 25-38 ◽

Cited By ~ 1

Author(s):

Brett Bowman ◽

Mincheol Kim ◽

Yong-Joon Cho ◽

Jonas Korlach

Keyword(s):

Dna Sequencing ◽

Real Time ◽

Single Molecule ◽

Long Read

Download Full-text

Steve Turner on "Single Molecule Real Time DNA Sequencing"

SciVee ◽

10.4016/11409.01 ◽

2009 ◽

Keyword(s):

Dna Sequencing ◽

Real Time ◽

Single Molecule

Download Full-text

De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

Scientific Reports ◽

10.1038/s41598-019-55908-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Joseph R. Fauver ◽

John Martin ◽

Gary J. Weil ◽

Makedonka Mitreva ◽

Peter U. Fischer

Keyword(s):

Single Molecule ◽

New Technologies ◽

Reference Genome ◽

De Novo ◽

Complete Mitochondrial Genome ◽

Nuclear Genome ◽

Brugia Malayi ◽

Field Isolates ◽

Sequencing Technologies ◽

Long Reads

AbstractFilarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.

Download Full-text

Five Closed Salmonella enterica Genome Sequences from a 2017–2018 Multistrain, Multistate Kratom Outbreak

Microbiology Resource Announcements ◽

10.1128/mra.01395-19 ◽

2020 ◽

Vol 9 (3) ◽

Author(s):

Hallie E. Rauch ◽

Julie Haendiges ◽

Maria Balkey ◽

Maria Hoffmann

Keyword(s):

Dna Sequencing ◽

Real Time ◽

Single Molecule ◽

Salmonella Enterica ◽

Genome Sequences ◽

Circular Chromosome ◽

Content Type ◽

Single Plasmid

We report here the closed genomes of Salmonella enterica strains from the 2017–2018 multistrain, multistate kratom outbreak using single-molecule real-time DNA sequencing. Four of the genomes consist of one circular chromosome, and the fifth has a circular chromosome and a single plasmid.

Download Full-text