Sanger sequencing

Drones or Unmanned Aerial Vehicles (UAVs) have huge potential to improve the safety and efficiency of sample collection from wild animals under logistically challenging circumstances. Here we present a method for surveying population health that uses UAVs to sample respiratory vapor, 'whale blow,' exhaled by free-swimming humpback whales (Megaptera novaeangliae), and coupled this with amplification and sequencing of respiratory tract microbiota. We developed a low-cost multirotor UAV incorporating a sterile petri dish with a remotely operated 'blow' to sample whale blow with minimal disturbance to the whales. This design addressed several sampling challenges: accessibility; safety; cost, and critically, minimized the collection of atmospheric and seawater microbiota and other potential sources of sample contamination. We collected 59 samples of blow from northward migrating humpback whales off Sydney, Australia and used high throughput sequencing of bacterial ribosomal gene markers to identify putative respiratory tract microbiota. Model-based comparisons with seawater and drone-captured air demonstrated that our system minimized external sources of contamination and successfully captured sufficient material to identify whale blow-specific microbial taxa. Whale-specific taxa included species and genera previously associated with the respiratory tracts or oral cavities of mammals (e.g., Pseudomonas, Clostridia, Cardiobacterium), as well as species previously isolated from dolphin or killer whale blowholes (Corynebacteria, others). Many examples of exogenous marine species were identified, including Tenacibaculum and Psychrobacter spp. that have been associated with the skin microbiota of marine mammals and fish and may include pathogens. This information provides a baseline of respiratory tract microbiota profiles of contemporary whale health. Customized UAVs are a promising new tool for marine megafauna research and may have broad application in cost-effective monitoring and management of whale populations worldwide.

Background: The availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2 kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches.

Background
GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.

Methods
We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.

Results
The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%,  respectively. We also found 92.4%overall agreement of two
molecular methods for the detection of RIF susceptibility. A  total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert.
Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.

Conclusion
Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant
isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

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.