MinION@Cadagno – Exploring the functional potential of the Lake Cadagno microbiome through nanopore sequencing, a pilot study
Nicole Liechti, Corinne P. Oechslin, Matthieu Bueche, Samuel M. Luedin, Francesco Danza, Nicola Storelli, Joël F. Pothier, Christian Beuret, Matthias Wittwer and Mauro A. Tonolla
Unpublished pilot study
Statement of contribution
I contributed to sampling, sample preparation, DNA extraction and data interpretation and manuscript preparation.
Research objective
The aim of the study was to test for the technical feasibility of future nanopore based metagenomic sequencing of the complex microbial community of Lake Cadagno under field conditions.
5.2.1 Introduction
The microbial community of Lake Cadagno (Ticino, Switzerland) has been studies for decades and represents an ancient ocean homologue due to its permanent stratification. It is situated in the Swiss Alps at about 2’000 meters above sea level [122]. Bottom water rich in sulfide provides the living condition for a dense population of mostly anoxygenic photosynthetic bacteria, belonging to the families of Chromatiaceae and Chlorobiaceae [154]. However, the microbial population has only been classified by 16S rRNA gene sequences [143]. This allows only a limited insight into the complex community with a taxonomic resolution to the genus level. As with the sequencing decreasing cost and ease of DNA sample preparation more bacterial ecosystems are studied using meta-omics. Thereby,
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the functional potential can be studied, as well as insight into phylogeny and evolution of microbial communities can be gained in unseen resolution.
The recent development of nanopore-based sequencing techniques, fast library preparation protocols, as well as hard- and software enables on site genomic studies with minimal lab equipment [431]. The Research Center in the Piora valley provides an ideal training ground to do nanopore-based metagenomics in a remote, minimal lab setting. As we expect the autumnal community shift in the interphase (chemocline at 12–14 m depth) from the dominating large celled Chromatium okenii (up to 105 cells ml-1) to small celled aggregated Thiodictyon and Lamprocystis type PSB in September, we plan to take samples then to obtain a profile from a population with a larger beta-diversity. This may help to further disentangle the different biochemical cycles present in Lake Cadagno. With this study we tested feasibility of on-site sequencing at Lake Cadagno.
5.2.2 Methods
Experiments were performed at the Alpine Biology Center in Piora located at 2’000 m a.s.l.
The field lab was set up in a simple field lab equipped with a centrifuge for 50 ml tubes and 1.5ml Eppendorf tubes as well as -20 °C and 4 °C fridges, water bath and heat block (Figure 5.2-1). In Mountain areas of this elevation the average day temperature is around 10 °C and drops to 0 °C during night in September. Therefore, low temperatures may affect experimental conditions and slow down the processes of sequencing library preparation. The conditions for the sequencing process are then controlled by the MinIon device.
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Figure 5.2-1 | Overview on the minimal lab equipment used for sample preparation, DNA extraction, library preparation and on-site sequencing in this study
DNA extraction and purification
For cell lysis 200 µl AVL buffer (Qiagen, Switzerland), 20 µl Proteinase K, 4 µl RNaseA (Qiagen) as well as approximate 25mg Lysozyme was added. After incubation for 5 min at room temperature (warmed by hand) cells were lysed by incubating at 56°C for 30 min. DNA was extracted using Qiagen DNeasy Blood and Tissue kit (Qiagen) according to manufactures protocol performing two AW1 washing steps and elution of the DNA in to steps using 100 µl prewarmed nuclease free water. The amount of DNA was measured using the dsDNA HS assay 2.0 on a Qubit fluorometer (Thermo Fisher). To concentrate the DNA, sample was purified using Agencount AMPure Beads (Beckmann, Coulter) according to manufactures protocol and eluted in 50 µl water.
DNA library preparation and MinION sequencing
Nanopore sequencing library was prepared using Rapid Sequencing Kit RAD003 (Nanopore, Oxford, UK) with a total DNA input of 200 µg in 7.5 µl. For the reaction 1.5 ml DNA low bind Tubes (Eppendorf, Switzerland) was used. Tagmentation step was performed for 2 min at 30 °C in water bath and inactivation step at 80 °C for 2 min using a heat block. Prior to
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sequencing, the Flow Cell QC was conducted, and the number of active pores was examined.
Finally, the library was loaded to the SpotON Flow Cell R9.4 and sequenced for 48h. As temperature in the mountain areas at time point of sampling drops to around 0°C during the night, the MinION device was enclosed in a Styrofoam box to prevent gadget from cooling during sequencing run.
Binning & Assembly
‘Live’ base calling was performed during sequencing using MinKNOW(version). Poretools v0.6.0 [432] (Loman and Quinlan, 2014) was used to extract reads in fastq format from base called fas5 files and to perform basic statistical analysis. For the taxonomic classification of the reads, de novo assembled genomes of C. okenii str. LaCa str. Cad16T, and str. CadA31 were added to the NCBI nr database (accessed 2018). Reads with a minimal length of 500bp were then classified using Centrifuge [433]. Output files were visualized using Pavian [434], an R based application for visualizing metagenomic datasets. In a second step, reads were aligned to the same database (NCBI nr + 3 additional genomes) using blastn (default parameters) [435]. Further, reads were mapped to the species of interest (C. okenii, str.
Cad16T, str. CadA31) using bwa mem specifying -ont option for nanopore data [436].
5.2.3 Results
Sequencing on MinIon during 48 h resulted in a total of 40’017 reads with a mean read length of 3’336 bp. For further analysis reads shorter than 500 bp were removed, resulting in 23’531 reads for downstream analysis. By using centrifuge in combination with the NCBI nt database with 3 additional de novo assemblies originated from Lake Cadagno bacterial strains, a total of 19’003 reads could be taxonomically classified. Out of them, 11’979 are considered as Eukaryotic sequences while 6’697 belong to Bacteria, 36 to Archaea and 88 to viral species (Figure 5.2-2). The PSB Chromatium okenii dominates the community whereas members of the class Actinobacteria, α- and β- proteobacteria are also abundant. The dominance of C.
okenii findings is in accordance with the flow cytometry based counting in August 2017 [134].
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Figure 5.2-2 | Taxonomic tree of the Lake Cadagno metagenomic chemocline sequences after centrifuge classification using the NCBI nr database and the complete genomes from PSB str. Cad16T, CadA31 and C. okenii LaCa. Numbers indicate the reads allocated for each taxonomic level. Scale indicates the hierarchical taxonomic level from left to right; D: Domain, P:
phylum C: class, O: order, F: family, G: genus and S: species.
5.2.4 Discussion
We showed the feasibility of metagenomic sequencing under field conditions at a simple laboratory setting at 2’000 m.a.s.l. However total DNA concentration was low and possible contaminations with eukaryotic occurred. The sample composition might has been biased by the centrifugation step, thereby enriching for large celled PSB C. okenii. Therefore we plan to repeat the experiment in September 2018 with an improved sampling protocol involving tangential flow filtration (TFF), CTD measurements and FC, DAPI and FISH enumeration of the samples. Salt free extraction buffers possibly improve protease and RNAase performance during DNA purification. As taxonomic classification is dependent on the database, we will
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include also a dataset with illumina based metagenomic bins from 2013/2014 (Petra Pjevac, University of Vienna, personal communications)
Acknowledgements
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ACKNOWLEDGEMENTS 6
…Professor Mauro Tonolla for giving me the opportunity to work on this project that he constantly fueled with heartily support, fruitful inspiration and trust. He gave me all the scientific freedom needed to explore Lake Cadagno in-depth.
…Dr. Michel Goldschmidt and Prof. Dr. Michael Hothorn to for serving in my PhD advisory committee and supporting me throughout the years with scientific advice.
…Dr. Matthias Wittwer for his constant passion, lavish support, for scientic advice and humor during all these years. The many fruitful and insightfuly discussions about my project with him hepled me to bring it further an opend up new possibilities.
…Dr. Joël F. Pothier ZHAW for introducing me into the many use of informatics in microbiological research and the constant support in my project and as a member of the advisory committee and beyond.
…Dr. Nadia Schürch for her belief in this project and substantial support.
…Dr. René A. Brunisholz for his commitment to science and helping me hands-on in developing and exploring an LC-MALDI platform. Without his valuable firsthand experience and his humorous wit I would not have made that far.
…my co-workers at the LMA in Bellinzona, Francesco Danza, Nicola Storelli, Michela Ruinelli, Samuele Roman, Federica Mauri, Damiana Ravasi, Sophie de Respinis and Andreas Bruder for their collaboration, support and the many Schneeberger for their interest in my project, their support and teaching and the many funny and cheerable moments.
…Sandra Paniga, Susanne Thomann and Fritz Wüthrich for taking care of all the supplies and equipment.
…Werner Arnold, Marc Avondet, Christian Beuret and all others for their constant support in Spiez.
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…the CBA foundation and the numerous guardiani for their valuable support in field work.
…Valentin Pflüger who is constantly and passionaly linking, inspiring and supporting the MALDI research community and of whom I profited a lot in these past years.
…Dottores Matthieu Bueche and Bernard Jenni thanks to whom I ‘discovered ‘ that there are sulfurous sources in the Jura and a lot af fascinating protist around in every pond.
…Dr. Jakob Zopfi, Prof. Pilar Junier and Dr. Anupam Sengupta for their advice and constant interest in this project.
…Professor Nils-Ulrich Frigaard and Dr. Yonghiu Zeng from the University of Copenhagen for the many discussion and insightful comments on my work and for sharing information and data on Lake Cadagno.
…Petra Pjevac and Jasmine Berg who were readily sharing data and with whom I could discuss metagenomics
…Tobias Sommer, Michael Plüss and Oscar Sepùlveda Steiner who gave me hands-on support on the platform and provided valuable CTD data and discussion during field work in 2015 to 2017.
…Lisa Eymann and Roman Lehner for their critical external perspective on my project and companionship as humble scientists.
…my friends with whom I could share the marvelous Piora experience on two
‘expeditions’ that were always interested in the research.
…my parents that always supported me a share the compassion an interest in nature
…Jenni who brought purple bacteria to dance!