MinION™ allied with 3D printed iChip – a workflow designed to let anyone discover new bacterial species in days
In this study, we adapt a protocol for the growth of previously uncultured environmental bacterial isolates, to make it compatible with whole genome sequencing. We demonstrate that in combination with the MinION sequencing device, complete assemblies can be derived, allowing genomic comparisons to be made. This approach allows rapid, inexpensive and straightforward discovery, and genomic analysis, of previously uncultured prokaryotic genomes, and brings greater ownership of all parts of the sequencing process back to individual researchers.
In addition to the broad variety of applications for the MinION developed by the MinION Community, Oxford Nanopore Technologies Ltd. and Metrichor Ltd. have an Applications group that spans Oxford and New York City. We are pleased to present this pre-print describing work that we have done to develop a method to allow easy and accessible whole genome sequencing and complete de novo assembly of ‘unculturable’ bacteria by modifying a way to grow these bacteria and combining it with our MinION sequencer.
The process of discovering new bacterial species can be time-consuming, costly, and cumbersome. Efforts to sequence bacterial genomes has consequently largely been limited to serious pathogens, such as Mycobacteria,Salmonella, and Streptococcus.
However, it is estimated that there are hundreds of thousands, or even millions, of different bacterial species, living in almost every environment on earth. These have many potential uses, for instance in biotechnology for biofuel production or the development of new antibiotics. Many environmental bacteria are unable to grow in standard laboratory conditions because they have special nutritional requirements only found in their natural setting.
A recently-published way to trick these unculturable bacteria to grow in culture is by using an iChip 1. This is a small 3D printed plastic device that allows for selective growth of individual bacteria in their natural growth medium, which could be river or ocean water, sewage or fresh water from distribution pipes, or even bodily fluids like blood, urine, or saliva.
In this workflow, we culture these otherwise unculturable bacteria and we extract the DNA and prepare sequencing libraries. This analysis workflow can be completed in a morning. Data from nanopore sequencing of the bacterial genome on a MinION is streamed in real time, with real time basecalling. This allows species identification in real time using analysis tools provided by Metrichor.
The long reads available from a MinION sequencer, which processes any length of DNA fragment presented to it and will routinely process reads tens of kilobases in length, greatly simplify the process of de novo genome assembly. This is true even if the genome is highly repetitive in nature.
To demonstrate this process, we applied the iChip to culture environmental bacteria present in the stream behind our laboratories in Oxford. We then grew the bacterial cultures further using a growth medium consisting of stream water supplemented with various nutrients. We extracted the genomic DNA from the bacteria, sequenced it on the MinION and de novo assembled the genomes. We are presenting two of these bacterial genomes in this paper. One turns out to be Klebsiella Oxytoca, which is commonly found around plant roots, but a strain not previously sequenced. The other is possibly from the family Pseudomonas, but it has only 73% similarity to any bacterial genome in the NCBI database.
There remain several more types of bacteria from this experiment which we have sequenced and are currently analysing. Our plan is to find collaborators to help correlate the genomic information with live-bacteria phenotypes to understand these new strains in more depth, and to see if they be utilized for biotechnological purposes. We also plan to use this combination of iChip and MinION sequencing to explore other environments, and possibly to investigate the effects on ecosystems resulting from environmental changes.
Lastly, as we continue to develop workflows that take advantage of the single-molecule nature of nanopore sequencing on a MinION, we aim to avoid the culturing steps altogether, to sequence and assemble multiple bacteria directly from a sample. We anticipate that this kind of assay will have a significant impact on the way bacterial infections are diagnosed, especially where the infectious bacteria are unculturable under laboratory conditions, where misdiagnoses are not uncommon. Coincidentally, this weekend a group from the MinION Access Programme published findings on monitoring urinary tract infections quickly using the MinION.
We developed this workflow with our goal in mind – to enable any person to do any biological analysis, in any environment – a goal that mirrors the low hardware, cost and IT requirements of our technology. We believe this workflow is a subject that might be of interest to individual researchers or eventually educators, people in industry/environment or even citizen science participants.
- Nichols D, et al. Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Applied and Environmental Microbiology 76 (8), 2445-2450 (2010)