Minknow

FASTQ files were generated for each run using MinKNOW (version 18.2.9; Oxford Nanopore Technologies Ltd.) and demultiplexed with Porechop (32 (link)) and qcat (version 1.01; Oxford Nanopore Technologies Ltd.). Reads were mapped to the M. tuberculosis H37Rv reference strain (GenBank accession number AL123456.3) with minimap2 (33 (link)), and consensus sequences and variants for each sample were generated using samtools (25 (link)) and custom in-house Perl scripts (available on request). Alignments were inspected using AliView (34 (link)), and statistical analysis was performed in R (35 ).

500 ng of polyA-selected RNA was used as directed in the Direct RNA Sequencing Kit (SQK-RNA001, Oxford Nanopore Technologies). The library was loaded onto a minION (MIN-101B, Oxford Nanopore Technologies) with an R9.4.1 flow cell (FLO-MIN106, Oxford Nanopore Technologies). MinKNOW (v1.10.23, Oxford Nanopore Technologies) was run without live base calling for 48 hours.
Bases were called from fast5 files with the Albacore script read_fast5_basecaller.py (v2.1.10, Oxford Nanopore Technologies). 491,142 reads were sequenced. Reads were aligned to the genome with minimap2 (v2.9-r720; Li, 2018 (link)) using options –ax splice –k14 –uf. Bam files were visualized directly in IGV.

Data from Flowcell #1 was locally basecalled by offline MinKNOW (v18.12.9) (Oxford Nanopore, Oxford, UK) during the run on a Dell XPS 13 i7 laptop with 16 GB RAM and 500 GB SSD. Data from Flowcell #2 was basecalled offline after the run using Guppy (v3.0.3) (Oxford Nanopore, Oxford, UK). FASTQ files from both sequencing runs were concatenated and uploaded to Kaiju (kaiju.binf.ku.dk) for taxonomic classification upon return to civilisation due to code errors running Kaiju offline in situ. Reads were compared to the “NCBI BLAST nr + euk” reference database using the “Greedy” setting, allowing mismatches with standard parameters (minimum match length 11, minimum match score 75, allowed mismatches 5).

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.

For ONT sequencing, a MinION sequencing library was prepared using the Nanopore Ligation Sequencing kit (Oxford Nanopore Technologies, Oxford, UK) as per the manufacturer’s protocol with starting DNA amount of 6 μg (185 fmol). A final amount of 650 ng (20 fmol) of the prepared library was loaded on a MinION Spot-On flow cell (Oxford Nanopore Technologies; version FLO-MIN106D R9.4.1) using a flow cell priming kit (Oxford Nanopore Technologies) as instructed by the manufacturer. The library was sequenced using a MinION device (Mk1C, MC110367) for 5 to 6 h with default instrument settings. Primary acquisition of data and real-time base calling was carried out using the graphical user interface MinKNOW (version 20.10.6; Oxford Nanopore Technologies) and Guppy base caller (v4.5.2; Oxford Nanopore Technologies).

FASTQ files were generated for each run using MinKNOW (version 18.2.9; Oxford Nanopore Technologies Ltd.) and demultiplexed with Porechop (32 (link)) and qcat (version 1.01; Oxford Nanopore Technologies Ltd.). Reads were mapped to the M. tuberculosis H37Rv reference strain (GenBank accession number AL123456.3) with minimap2 (33 (link)), and consensus sequences and variants for each sample were generated using samtools (25 (link)) and custom in-house Perl scripts (available on request). Alignments were inspected using AliView (34 (link)), and statistical analysis was performed in R (35 ).

500 ng of poly(A)-selected mRNA was used as input for the Direct RNA Sequencing Kit (SQK-RNA002, Oxford Nanopore Technologies), used as directed with a modified reverse transcription (RT) step. Marathon reverse transcriptase (kindly gifted from Dr. Kathleen Collins) was used for the RT instead of Superscript III. The RT reaction was performed in 1X first strand buffer (20 mM Tris-HCl pH 7.5, 75 mM KCl, and 5 mM MgCl₂), with 0.8 mM dNTPs, 8 mM DTT, and 20 μM Marathon reverse transcriptase. The RT reaction was incubated at 37°C for 50 min then 70°C for 10 min. Downstream steps were followed according to kit instructions. The library was loaded onto an R9.4.1 flow cell (FLO-MIN106, Oxford Nanopore Technologies) and sequenced on a minION (MIN-101B, Oxford Nanopore Technologies). MinKNOW (v22.05.5, Oxford Nanopore Technologies) was run without live base calling for 72 hours. Bases were called from fast5 files using Guppy (v6.0.1, Oxford Nanopore Technologies). Reads were aligned to the S288C reference genome (SacCer3) using the EPI2ME Desktop Agent (v3.5.6, Oxford Nanopore Technologies). Bam files were visualized directly in Integrated Genomics Viewer (IGV, Broad Institute).