Minion flow cell r9

The ligation sequencing kit (SQK-LSK109, Oxford Nanopore Technologies, Oxford, UK) was used and then real-time sequencing technique, MinION (Oxford Nanopore Technologies, Oxford, UK) was used (30 (link)). Briefly, DNA was extracted with Magnetic Soil and Stool DNA Kit (Tiangen, China). The concentration of the extracted DNA (∼1 μg) was measured with Qubit fluorometer (Thermo Fischer Scientific, Waltham, MA, USA). For fragmented DNA repair and end-repaired DNA, NEBNext FFPE repair Mix and NEBNext end repair/dA-tailing Module (New England BioLabs Inc., Ipswich, MA, USA) was used, respectively. After DNA purification with AMPure XP beads (Beckman Coulter, Brea, CA, USA), the sample was loaded on the MinION Flow Cell R9.4 (Oxford Nanopore Technologies, Oxford, UK). Sequencing protocol was applied using the nanopore sequencing software, MinKNOW (v1.10.23, 2017, Oxford Nanopore Technologies, Oxford, UK), in order to collect electronic signal data. After basecalling of long reads with Oxford Nanopore software GUPPY 3.0.3 (to convert fast5 files in fasta format), each output file from the nanopore sequencing was BLAST searched against the “nucleotide collection (nt)” database from NCBI. Complete BLAST outputs for each sample were imported into MEGAN v6.21.12 using the default parameters.

The complete sequence of plasmid was determined by sequencing using long-read MinION (Oxford Nanopore) and short-read Illumina MiSeq technologies. Plasmid DNA was extracted from transformants with the Qiagen Plasmid Midi Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions and then sent to Biomarker Technologies for sequencing (Biomarker, Beijing, China). The long reads were generated by Oxford Nanopore MinION flowcell R9.4 (Li et al., 2018 ) with a depth more than 300 times and the 150 bp paired-end short reads were generated by Illumina MiSeq system (Illumina, San Diego, CA, United States) with a depth more than 100 times. The MinION reads were filtered using Filtlong (version 0.2.0) to remove any reads <2000 bp, followed by removal of the lowest 10% of reads by quality. Complete sequence assembly was performed with Canu version 1.5 (Koren et al., 2017 (link)) using a combination of short and long reads, followed by error correction by Pilon version 1.12 (Walker et al., 2014 (link)). Gene prediction and annotation were done with RAST,² and IS finder.³ Plasmid replicon types and the plasmid MLST (pMLST) were analyzed using the CGE server.⁴ Sequences were analyzed and compared using BLAST,⁵ and map generation was performed using Easyfig (version 2.3) (Sullivan et al., 2011 (link)) and BRIG (Alikhan et al., 2011 (link)).

The metagenome of the green-turquoise mat was sequenced using a 1D ligation sequencing kit (SQK-LSK108). Sequencing was performed using an Oxford Nanopore Technologies (ONT) MinION flow cell R9.4 containing an initial number of 1’553 active nanopores for a duration run of 48 hours, using the MinKNOW software (v18.01.6). The ONT Guppy basecaller (v2.3.7) was further used to assign base names on the resulting chromatogram.