The phyllosphere of Euphorbia lateriflora and Ficus thonningii
The phyllosphere, which includes the microbiome of the leaves of plants, remains an understudied ecological niche. The bacterial colonizers of medicinal plants have barely been studied even though their host plants have been used widely in ethnomedicine. Part of the limitations of studying phyllosphere bacteria is that they cannot be sufficiently classified using traditional culture and biochemical methods, largely because such methods were created with a focus on medically important bacteria which represent only a small fraction of the group. Consequently, phyllosphere studies conducted before the era of genomics were mostly species in-specific or focused on bacterial pathogens/symbionts of food plants. It soon became clear as phyllosphere studies progressed that some organisms, including novel ones, were missed as they were not covered even in the classic Bergey’s manual of systemic bacteriology database. With the advent of sequencing, it has become possible to study phyllosphere bacteria in detail, both culturable and unculturable. Metagenomic DNA can be obtained directly from plant colonizers and sequenced directly. Phyllosphere studies currently employ sequencing to characterize bacteria, including novel species. We used sequencing to characterize phyllosphere bacteria of Ficus thonningii and Euphorbia laterifolia which are Nigerian medicinal plants, hypothesizing that both plants are colonized by diverse bacteria which are advantageous. Bacteria were isolated on 0.1X tryptone soya agar, after which isolates were identified by 16S rRNA gene sequencing, compared by multiple sequence alignments and phylogenetics. Select isolates were further classified biochemically with Microbact 24E and genotypically by whole genome sequencing. A total of 100 and 77 isolates from F. thonningii and E. lateriflora respectively were identified by 16S rRNA sequencing. The dominant phylum and family from both plants were Proteobacteria and Enterobacteriacea respectively. 12 other bacterial families were encountered. Biochemical and genomic classification of 14 isolates which could not be assigned to any species based on 16S rRNA analyses were discordant as Microbact identified all but two as Acinetobacter, while genome sequencing classified them as Brevibacteria, Agrococci, Kocuria and others. Plasmids, resistance and virulence genes were also detected in a sub set of isolates. Study of phyllosphere bacteria was greatly limited by traditional microbiology methods up until introduction of sequencing into the field. Apart from correctly identifying organisms, sequencing provides extensive insight into phylogeny and metabolic capacity of organisms, making it possible to predict functions of the bacteria to host plants after a single sequencing experiment. This has greatly changed the ways in which the phyllosphere microbiome is studied.
Anderson O. Oaikhena is a Doctoral student and teaching assistant in the Department of Pharmaceutical Microbiology at the University of Ibadan. He is currently studying bacterial colonizers of medicinal plants, with a particular interest in colonization of antibacterial plants as a framework for better understanding the ecology of antimicrobial resistance in nature. Anderson’s outstanding performance in the Master of Science degree in Pharmaceutical Microbiology in 2017 earned him a doctoral scholarship award from the postgraduate college of University of Ibadan. He serves as a research associate in the Nigeria AMR surveillance network, where he is the point person for whole genome sequencing of antibiotic resistant bacteria.
Small, modified and highly structured: the challenge of tRNA sequencing
Oxford Nanopore Technologies have been initially developed to directly sequence the long molecules of DNA and RNA. The possibility of sequencing shorter molecules using nanopore is widely discussed in the field but remains questionable because of the higher error rate compared to the classical deep-sequencing approaches. Here we show the successful application of the MinION device to sequence tRNA molecules. The challenges of sequencing tRNAs are due its short length and folded structure, however, to overcome this we have improved the library preparation in order to compute the whole length of the tRNA. Initially, we sequenced a mixture of in vitro transcribed E. coli tRNAs and developed a bioinformatical pipeline to assign base-called reads to different tRNA species with a high degree of accuracy. Moving towards more complex samples containing native tRNAs, we found that modifications along the tRNA reduced the fidelity of called bases, so we developed an algorithm of tRNA classification based on raw-signal patterns. Comparing those patterns to unmodified in vitro transcribed tRNA signals allowed us not only to distinguish between different tRNA species, but also to detect modifications occurring in the native tRNAs. Our results show that nanopore-based approaches can be used to sequence tRNAs and classify them. This unveils a new area of the nanopore technology in application to short molecules, detecting the modifications and even predicting the potential ones, which are currently unknown, but may govern the structure, affect decoding or play a role in diseases.
Irina studied Medical Cybernetics at the Russian National Research Medical University in Moscow and graduated with a Medical Degree. She then completed a Masters in Biochemistry and Bioinformatics at the University of Potsdam in 2015, where she began working with NGS data. Irina is currently a PhD student at the Institute of Biochemistry and Molecular Biology at the University of Hamburg. As a bioinformatician, Irina processes various deep sequencing data including nanopore and develops new algorithms for analysis.
Biological evidence of the future: the use of sequencing in forensic DNA analysis
Forensic DNA profiling uses short tandem repeat (STR) analysis for human identification purposes, i.e. to establish a link between biological evidence and an individual. This technique is currently limited to assessing the length of STR alleles via capillary electrophoresis and relies on the comparison to a reference DNA profile. The advent of DNA sequencing has revolutionised the field of forensic genetics. Alleles with the same length but a different sequence can be distinguished, providing additional discrimination between individuals which can greatly aid in DNA mixture interpretation. Rare sequence mutations can be identified to differentiate identical twins, who cannot be told apart using conventional DNA profiling. Using sequencing, scientists have also begun to harness intelligence-based information that a biological sample can provide which could be of use in an investigation. The analysis of single nucleotide polymorphisms (SNPs) offers new opportunities in the form of forensic DNA phenotyping and forensic epigenetics. Prediction of eye, hair and skin colour, as well as bio-geographic ancestry and chronological age estimations of an unknown individual are all now possible. The introduction of nanopore sequencing technology has the potential to transform the field of forensic genetics even further. The portability and real-time capability of the MinION could shift analysis out of the lab into the field, greatly reducing cost and turnaround time which are critical in an investigation. Research into the feasibility of this technology for forensic applications is currently underway. Sequencing has not only changed the field of forensic genetics, but also has changed the way biological evidence is approached and could be used in investigations which has had a wide-reaching effect in enforcement, legal, governmental and judicial fields. Although not routinely used in forensic casework at present, many forensic laboratories around the world are currently validating sequencing technologies with the expectation that this will be the biological evidence of the future.
Rebecca Richards is a doctoral student in the Forensic Science Programme at the University of Auckland. Her research focuses on the development and optimisation of DNA methylation markers for forensic applications, specifically identical twin differentiation and chronological age estimation. Rebecca is also a senior technician in the Forensic Biology Group at the Institute of Environmental Science and Research (ESR), a Crown Research Institute which provides forensic services to the New Zealand Police. In addition, she is running point for the MinION research currently being undertaken at ESR and is involved in the wider validation of DNA sequencing for forensic use.