Results of the Cambridge Metagenomics Challenge Workshop July/August 2019 conducted by PuntSeq and the Cambridge Biomakespace

We conducted metagenomic analyses of a freshwater sample (400 ml) taken at the outlet of the wastewater treatment plant into the river Cam in Cambridge, East Anglia, UK. The sample was collected at 52.23201N, 0.17084E on August 4th, 2019. The sample was filtered through 0.22 µM pore-sized nitrocellulose membrane. We then performed DNA extractions with a modified DNeasy PowerWater protocol (Qiagen, Hilden, Germany). To apply both, shotgun sequencing and 16S rRNA targeted sequencing to the extracted DNA, we amplified and barcoded only a part of it for full-length 16S rRNA (~ 1.5 kb); in the end, we used 2000ng of shotgun DNA and 500ng of amplified DNA, which were pooled together and subjected to the Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit, to then be sequenced with a R9.4 MinION flow cell (Oxford Nanopore Technologies). The MinION instrument was run for approximately 48 hours, until no further sequencing reads were obtained. We here provide the raw fast5 files, to allow users to choose the basecalling algorithms, and to apply their own method for demultiplexing targeted and shotgun reads. We encourage you to visit our website www.puntseq.co.uk for more details on the PuntSeq project, and to get more information about future workshops.