Ion xpress plus fragment library kit

A total of 200 μL of sucrose cushion concentrated phage were subjected to nucleic acid extraction using GeneJET™ Viral DNA and RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer instructions. The phage DNA concentration was measured using Qubit 3 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using Qubit^TM dsDNA HS Assay Kit according to manufacturer instructions. Libraries preparation of phage DNA was conducted using Ion Xpress^TM Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, the plate was prepared using Ion 520^TM Kit-OT2. Finlay, the libraries were loaded on the Chip of ion S5^TM System. Reads were trimmed and assembled into a whole genome using the PATRIC bioinformatics resource center online platform (https://www.patricbrc.org) (accessed on 15 October 2021) after checking reads quality using the FastQC software version 0.11.4 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) (accessed on 15 October 2021) (Babraham, Cambridge, UK).

As a first step in the preparation of the fragment library for WGS, phage DNA was randomly physically sheared using a Covaris S220 focused-ultrasonicator. The barcoded DNA fragment library with the target insert size of 250 bp was prepared using Ion Xpress^TM Plus Fragment Library Kit (Thermo Fisher Scientific) following the manufacturer’s guidelines with NucleoMag^® NGS Clean-up and Size Select (MACHEREY-NAGEL) bead cleanups in between the library preparation steps. Library concentrations were verified on Agilent 2100 Bioanalyzer using hsDNA assay. Sequencing of the resulting library was carried out on an Ion Proton using Ion PI^TM chip (Thermo Fisher Scientific).

The library preparation for Oxford Nanopore MinION and Thermo Fisher Ion PGM sequencing was performed as explained previously (Panthee et al., 2017a (link), c (link), 2018 (link)). Briefly, for ultra-long reads library, 1 μg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, United States) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, United States). The DNA was then ligated to the adapter using NEB Blunt/TA Ligase Master Mix (NEB). The library was purified using Agencourt AMPure XP (Beckman Coulter Inc) and reads were obtained using the 48-h protocol and live base-calling after loading the library to a primed FLO-MIN106 R9.4 SpotON Flow Cell. The 400 base-reads was prepared after fragmentation of 100 ng of the DNA using the Ion Xpress^TM Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, United States). The libraries were enriched in an Ion ^TM318 Chip v2 using Ion Chef (Thermo Fisher Scientific), and subsequent sequencing was performed in the Ion PGM System (Thermo Fisher Scientific).

Soil DNA extraction and Next-Generation-Sequencing of bacterial 16S hypervariable regions were performed according to Cuartero et al. (2022 (link)). Briefly, soil DNA was extracted from 1 g of soil (wet weight) using DNeasy Power Soil Kit (Qiagen). Quantity and quality of DNA were tested through Qubit 2.0 fluorometer (Invitrogen, Thermo Fisher Scientific, USA) and NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Ion TorrentTM Personal Genome MachineTM (PGM) was employed to amplify 16S hypervariable region using Ion XpressTM Plus Fragment Library Kit in combination with Ion XpressTM Barcode adapter (Thermo Fisher Scientific), the detailed process is described in Cuartero et al. (2022 (link)).

For 16S rRNA amplicon sequencing, 2 μL of DNA extracted from each sample was used to construct the libraries, and the Ion GeneStudio^TM S5 System (Life Technologies, Carlsbad, CA, USA) was used. For this purpose, the 16S hypervariable regions were amplified with two sets of primers, v2–4–8 and v3–6, 7–9, and the libraries were prepared by using the Ion 16S^TM Metagenomics Kit (Life Technologies) and the Ion Xpress^TM Plus Fragment Library Kit (Life Technologies). Libraries containing equal amounts of PCR products pooled with a barcode were prepared by using the Ion Xpress^TM Barcode Adapters Kit (Life Technologies). Then, these libraries were quantified by using the Ion Universal Library Quantitation Kit (Life Technologies). Next, 10 pM of each library was pooled and loaded on an Ion OneTouch™ 2 System (Life Technologies), which automatically performs template preparation and enrichment. Template-positive ion sphere particles were enriched with Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Invitrogen, Carlsbad, CA, USA) by using an Ion One Touch ES instrument. Finally, an Ion 520 TM chip (Life Technologies) was loaded with the samples on an Ion GeneStudioTM S5 System sequencer using the Ion 520™ & Ion 530™ Loading Reagents supplied in the OT2-Kit (Life Technologies).