The microbiome is the collective community of microorganisms that inhabit a specific environment, such as soil, water or within a given organism. In environmental substrates, the microbiome can play an important role in ecological processes such as nutrient cycling. Within living organisms, the microbiome can contribute to overall health, and its composition can even be diagnostic for certain disease states.
Sequencing technology allows us to characterize these microorganisms without the use of culturing methods, which tend to miss the majority of microbes which are not culturable. The list of substrates and settings that have a microbiome to study is practically endless, resulting in a broad range of projects designed with partner schools across the ʻĀina-Informatics Network - including a soil study by Farrington High School students at the loʻi kalo at Niuhelewai, pictured in the banner above. Some other examples are pictured below.
Waipahu HS student swabbing the biofilm on a hōʻawa leaf - which critically endangered kāhuli are known to graze on - within the Pahole Natural Area Reserve.
Kahuku HS student sampling the sediment at Loko Ea fishpond as part of an investigation into nutrient cycling dynamics within the pond.
Soil sampling with Lānaʻi HS students in a māla kalo at Pālāwai Farm to investigate the impact of various food and cover crops on soil health.
Sample collection and preservation
Microbiome samples target bacteria, archaea, fungi and even viruses, and are optimally processed using DNA extraction methods suitable for microorganisms with fortified cell membranes. These methods often include homogenization steps using bead beaters or other high speed agitators to break through tough membranes. For soils, the Qiagen PowerSoil Pro Kit and the Macherey-Nagel NucleoSpin Soil Mini Kit have yielded consistent results. For water samples, we recommend filtering through 0.22 um membranes, and then extracting the DNA from the membranes using kits designed for soil. All samples should be kept on ice upon collection and extracted as soon afterwards as possible.
Lab Protocols
HANDOUT: 16S Sequencing Roadmap
16S PCR (For Bacterial microbiomes)
ITS PCR (For fungal microbiomes)
Equipment and reagents: PCR thermocycler, Taq Premix (i.e. NEB LongAmp Taq MasterMix), ONT 16S Barcoding Kit, microcentrifuge
Gel electrophoresis
Equipment and reagents: Gel electrophoresis system, agarose, running buffer, gel stain, DNA ladder
library preparation & Sequencing
Coming soon…
Bioinformatics
For bacterial microbiomes, version 5.2.5 of EPI2ME Labs, Oxford Nanopore Technology’s in-house bioinformatics platform, has several classification options within the wf-16S workflow. Alternatively, the wf-metagenomics workflow enables reconstruction of metagenome-assembled genomes (MAGs) from larger datasets, which can provide resolution on unculturable bacteria and reveal functional information about the microbial community.
To allow for direct comparison between bacterial communities, assess sampling completeness (rarefaction) and take into account additional metadata such as environmental measurements, we have a custom tool available via our ʻIolani Bioinformatics Portal called EPI2MEviz.
Note: Please standby, an update to EPI2MEviz in currently underway.
