Megaruptor system

Library preparation and sequencing were performed at the GeT-PlaGe core facility, INRAe Toulouse, according to the manufacturer’s instructions “1D gDNA selecting for long reads (SQK-LSK109)”. At each step, DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA purity was assessed using the nanodrop (Thermofisher) and size distribution and degradation were assessed using the fragment analyser (AATI) High Sensitivity DNA Fragment Analysis Kit. Purification steps were performed using AMPure XP beads (Beckman Coulter).
Flow cell was run with 7 µg of DNA previously purified and sheared to 25 kb using the megaruptor system (diagenode). Calibration was performed using the Short Read Eliminator Family: SRE size XS kit (Circulomics) to deplete short fragments. A one step DNA damage repair + END-repair + dA tail of double stranded DNA fragments was performed on 2 µg of sample. Adapters were then ligated to the library. The library was loaded onto R9.4.1 revD flow cells and sequenced on the GridION instrument (Oxford Nanopore Technologies) at 0.025pmol within 72 h.

Library preparation and sequencing were performed at the GeT‐PlaGe core facility, INRA Toulouse, according to the manufacturer's instructions “1D gDNA selecting for long reads (SQK‐LSK109).” Aiming at covering the M. graminicola genome at >70× with long reads, sequencing was done on one ONT flowcell. Genomic DNA was purified using AMPure XP beads (Beckman Coulter). Eight micrograms of purified DNA was sheared at 20 kb using the megaruptor system (Diagenode). A “one‐step” DNA damage repair + END‐repair + dA tail of double‐stranded DNA fragments was performed on 2 µg of DNA. Adapters were ligated to the library that was then loaded (0.03 pmol) onto an R9.4.1 revD flowcell. It was sequenced on the GridION instrument for 48 hr. Final reads were base‐called using Guppy v.1.8.5‐1 (Oxford Nanopore).
After sequencing, adapters of raw ONT reads were trimmed using Porechop (Wick, 2019). Only reads with a Q‐score value greater or equal to 7 were selected using NanoFilt v.1.1.0 (De Coster, D’Hert, Schultz, Cruts, & Van Broeckhoven, 2018). Minimap2 (Li, 2018) was used to map long reads to the M. graminicola mitogenome (GenBank no. HG529223), and Samtools Fasta ‐ f 0x4 (Li et al., 2009) was used to sort out long reads that mapped to this reference.