Metagenomic monitoring of freshwater resources with long-read real-time sequencing - basecaller: Albacore

We conducted metagenomic analyses of freshwater samples (400 ml) from nine distinct locations (samples 1-9, with 9.1 and 9.2 as biological replicates from the same site) along an eleven kilometer trajectory of the river Cam in Cambridge, East Anglia, UK. This was done at three time points in 2018: on April 15th, June 17th, and August 19th.Samples were filtered through 0.22 µM pore-sized nitrocellulose membranes. We then performed DNA extractions with a modified DNeasy PowerWater protocol (Qiagen, Hilden, Germany). 400 ml of deionised water were subjected to the same workflow as negative control (N). DNA isolates were barcoded according to their location or negative control ID, amplified for full-length 16S rRNA (~ 1.5 kb) and complemented by a positive control (P) containing a defined microbial community standard which features eight distinct bacterial species (ZymoBIOMICS D6305, Zymo Research, Irvine, CA, USA). The resulting amplicons were then sequenced with one R9.4 MinION flow cell (Oxford Nanopore Technologies) per time point. The MinION instrument was run for approximately 48 hours, until no further sequencing reads were obtained. Raw fast5 files were recorded and locally base called using Albacore (version 2.3.1). Reads were demultiplexed and adapters trimmed using porechop (version 0.2.4). The only non-default parameter was '--check_reads' (set to 50,000 to increase the subset of reads to search for adapter sets). Next, we removed all reads shorter than 1 kb using filtlong (version 0.2.0).Here, we deposit the de-multiplexed and processed reads as one fastq file per time point and location (or control).We encourage you to visit our website www.puntseq.co.uk for more details on this project. Combined results from these sequencing runs will be released soon on bioRxiv - stay tuned!