From cheek swabs to consensus sequences : an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

Background: Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results: Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions: All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources

Transposing from the laboratory to the classroom to generate authentic research experiences for undergraduates.

Large lecture classes and standardized laboratory exercises are characteristic of introductory biology courses. Previous research has found that these courses do not adequately convey the process of scientific research and the excitement of discovery. Here we propose a model that provides beginning biology students with an inquiry-based, active learning laboratory experience. The Dynamic Genome course replicates a modern research laboratory focused on eukaryotic transposable elements where beginning undergraduates learn key genetics concepts, experimental design, and molecular biological skills. Here we report on two key features of the course, a didactic module and the capstone original research project. The module is a modified version of a published experiment where students experience how virtual transposable elements from rice (Oryza sativa) are assayed for function in transgenic Arabidopsis thaliana. As part of the module, students analyze the phenotypes and genotypes of transgenic plants to determine the requirements for transposition. After mastering the skills and concepts, students participate in an authentic research project where they use computational analysis and PCR to detect transposable element insertion site polymorphism in a panel of diverse maize strains. As a consequence of their engagement in this course, students report large gains in their ability to understand the nature of research and demonstrate that they can apply that knowledge to independent research projects

Incorporating molecular data in fungal systematics: a guide for aspiring researchers

The last twenty years have witnessed molecular data emerge as a primary research instrument in most branches of mycology. Fungal systematics, taxonomy, and ecology have all seen tremendous progress and have undergone rapid, far-reaching changes as disciplines in the wake of continual improvement in DNA sequencing technology. A taxonomic study that draws from molecular data involves a long series of steps, ranging from taxon sampling through the various laboratory procedures and data analysis to the publication process. All steps are important and influence the results and the way they are perceived by the scientific community. The present paper provides a reflective overview of all major steps in such a project with the purpose to assist research students about to begin their first study using DNA-based methods. We also take the opportunity to discuss the role of taxonomy in biology and the life sciences in general in the light of molecular data. While the best way to learn molecular methods is to work side by side with someone experienced, we hope that the present paper will serve to lower the learning threshold for the reader.Comment: Submitted to Current Research in Environmental and Applied Mycology - comments most welcom

DNA sequencing: bench to bedside and beyond†

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2)

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis

High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq

Salmonella enterica is represented by \u3e2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample. Poultry is a major reservoir of Salmonella spp., including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of Salmonella spp. in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003% of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel