Mnt 001

Library preparation was carried out using the Rapid Barcoding Sequencing Kit (SQK-RBK004, Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions. The finished library was loaded onto a R9.5.1 MinION flow cell (FLO-MIN107, Oxford Nanopore Technologies). Sequencing was conducted under the standard settings using a MinION (Mk1B, Oxford Nanopore Technologies) in combination with a MinIT (MNT-001, Oxford Nanopore Technologies). Signal information (fast5) was basecalled with MinIT and Guppy v2.1.3 (Oxford Nanopore Technologies).

Sequencing was conducted using the MinION with a Flongle flow cell (R9.4.1) and with a MinIT (MNT-001) (Oxford Nanopore Technologies, UK) for basecalling. At the time that this field expedition took place (summer 2021), the MinIT was available as a companion to the MinION but has since been discontinued. It can be replaced with a laptop or the Mk1b device for basecalling. The Flongle flow cell provided an adequate sequencing depth for 16S rRNA gene amplicon surveys at a very low cost (~$90 USD). Sequences were locally basecalled using MinKNOW (v 4.3.20) (Oxford Nanopore Technologies, UK), connected to a Dell Inspiron 13–7378 laptop with 16 GB RAM and 512 GB SSD (Dell, USA). The length of the sequencing runs was variable and depended on Flongle flow cell quality and desired number of sequences per sample. In general, runs were continued until the active pores in the flow cells were depleted. As an example, to obtain 5000 sequences per sample with six barcoded samples (i.e., 30,000 sequences total), using a Flongle with around half of the pores available (~60 pores), approximately 2 h of sequencing is required. On average, Nanopore amplicon libraries in this study contained 18,630 reads.

The Fast5 files generated during sequencing were basecalled to FastQ files by using Guppy v.3.4.5 (Oxford Nanopore Technologies) on the MinION IT device (MNT-001). Furthermore, barcoded reads were demultiplexed and identified barcodes were clipped. The reads separated by barcode were then further trimmed with Porechop³, removing residual barcode and adapter sequences and separating chimeric reads into separate reads. The trimmed FastQ reads for each sample were aligned to the used strain reference if known; for patient samples strain Wuhan-Hu-1 (Accession NC_045512) was used as a reference with Guppy v.3.4.5. Based on the resulting BAM files, primer sequences were soft clipped with bamclipper v.1.1.1 and all soft clippings from the BAM file were removed with custom python scripts. The assembled file was then polished and the consensus sequence was called using medaka v. 1.2.3⁴. Furthermore, the resulting consensus sequences were masked at regions with coverage depth <10, based on bedtools v2.26.0 genomecov histograms⁵ with custom python scripts. Ambiguities and indels in homopolymer regions were corrected manually, based on the reference genome. For each sample, 50,000 reads were analyzed.
All sequence data is deposited under the project number PRJEB42647 at the European Nucleotide Archive.