As part of the SMARTEn project we have performed several DNA sequencing experiments. As we expect that the data we generated will be useful to anyone working with MinION, below we provide details of how it was generated and how it can be downloaded.
We performed MinION sequencing of mock community consisting of five algal species and two bacterial species. The cultures were purchased from the University of Texas at Austin. The table below provides references.
| Name | Type | NCBI ID | UTEX ID | NCBI Tax ID |
|---|---|---|---|---|
| Chlamydomonas reinhardtii | Green algae | GCA_000002595.3 | UTEX 2243 | 3055 |
| Synechocystis sp. | Cyanobacteria | GCA_000478825.2 | UTEX B 2470 | 1147 |
| Anabaena flos-aquae | Cyanobacteria | GCA_000521175.1 | UTEX LB 2558 | 284502 |
| Chlorella vulgaris | Green algae | GCA_001021125.1 | UTEX 2714 | 3077 |
| Scenedesmus obliquus | Green algae | GCA_002149895.1 | UTEX 393 | 3088 |
| Nannochloropsis oculata | Algae | GCA_004335455.1 | UTEX 2164 | 1259847 |
| Euglena gracilis | Algae | GCA_900893395.1 | UTEX B 367 | 3039 |
The DNA extraction was performed using the FastDNA Spin Kit for Soil (MoBio, Solon, OH) following manufacturer instructions. DNA was eluted in 50µL warmed Elution Buffer. The extracted DNA was quantified using a QUBIT 3.0 fluorometer (Invitrogen), and mixed in differing proportions to create three unique mock communities. Mock community compositions were calculated based on the relative DNA mass contributed by each organism. The following mock communities were obtained:
The sequencing libraries were prepared using the Genomic DNA by Ligation sequencing kit SQK-LSK109 (Oxford Nanopore Technologies, Oxford, United Kingdom) and the corresponding protocol (Version: GDE_9063_v109_revX_14Aug2019). As recommended, a minimum of 1µg of genomic DNA was utilized for sequencing library preparation and no additional fragmentation steps were performed.
Prepared sequencing libraries were loaded onto a FLO-MIN106D flow cell (R9.4.1 chemistry) and sequenced using a MIN-101B MinION sequencing device. Sequencing was performed using a 48-hour run script with active channel selection enabled and refueling was performed at approximately 22 hours by loading 250µL Flush Buffer (FB) into the flow cell.
Raw MinION reads were basecalled using Guppy 5.0.7+2332e8d.
Currently, all data is available from our Box cloud service. Please use the following link to access the data: https://buffalo.box.com/s/7p5uu1j1ldekkvzw0qqm0bb8gep8xm2v
To test our metagenomic classifier we used a reference database consisting of the key Algae and Cyanobacteria reference genomes combined with an additional set of bacterial genomes. All reference sequences came from the NCBI RefSeq. Below we provide a collection of files that can be used to reconstruct our reference database: https://buffalo.box.com/s/0nhldjstw3cfxvg2elh4yieinc5z3416