Nebnext ffpe repair mix

We constructed a genomic library for ONT sequencing using the ONT 1D ligation sequencing kit (SQK-LSK108) according to the manufacturer’s instructions^{8 (link),9 (link)}. We constructed the library in three steps and measured the DNA concentration using a PicoGreen assay at the end of each step (Table 1). First, we subjected 2.0 μg of genomic midge DNA to DNA repair using an NEBNext FFPE Repair Mix (NEB cat no. M6630) to eliminate DNA fragmentation. After purification using AMPure XP beads, we subjected the repaired genomic DNA to end repair and dA-tailing using an NEBNext Ultra II End-Repair/dA-tailing Module (NEB cat no. E7546), and purified the DNA using AMPure XP beads. We ligated an adapter for sequencing to the purified DNA using adapter mix 1D in an SQK-LSK108 kit and an NEB Blunt/TA ligase Master Mix (NEB cat no. M0367). Finally, we cleaned-up the adaptor-ligated DNA using AMPure XP beads, an ABB buffer, and an elution buffer. We quantified the final library using a Qubit.

Yeast cells were grown in 50 ml of YPD to a concentration of >10⁷ cells/ml. The methods of library construction and Nanopore sequencing were described in [51 (link)]. Briefly, input DNA was treated with NEBNext FFPE RepairMix (M6630; NEB) to repair single-strand nicks, then DNA ends were repaired to form blunt ends using NEBNext End Repair Module (E6050; NEB). Samples were purified using AMPure XP beads. Each sample was barcoded using the Oxford Nanopore native barcoding genomic DNA kit (EXP- NBD104), followed by adapter ligation using ligation-based library kit (SQK-LSK109). After final purification, multiple barcoded libraries were loaded onto MinION flow cells (FLO-MIN106D R9.4.1) and analyzed on a MinION sequencer (MIN-101B).
The monitoring of the sequencing process, as well as real-time basecalling, were performed using Minknow (v21.02.1). The build-in basecaller (Guppy,4.3.2) was used to simultaneously convert raw electrical signals into fastq files. Output fastq files were aligned to S288c genome (sacCer3) using NGMLR [52 (link)]. The resulting.sam files were converted to.bam files using Samtools [45 (link)]. The sorted bam files were used to call structural variants by Sniffles [52 (link)] generating.vcf files. Both sorted bam file and.vcf file were uploaded to Ribbon [36 (link)] to visualize genomic structural variants.

Approximately 1 μg of native genomic DNA or purified LR-PCR amplicons were prepared for ONS sequencing on R9.4.1 flow cells using the Ligation Sequencing Kit SQK-LSK109 (Nanoporetech), in combination with the Native Barcoding Expansion Kit EXP-NBD114 (Nanoporetech). Genomic DNA was fragmented either through BamHI digestion (Materials and Methods) or sheared to 10 kb using g-tubes (Covaris), following manufacturers’ instructions. Simultaneous DNA repairing, end-repairing and dA-tailing was achieved using the NEBNext FFPE Repair Mix (NEB) and the Ultra II end-repair module (NEB). Barcodes were ligated to individual samples using Blunt/TA Ligase Master Mix (NEB). Samples were then combined and AMII adapters containing the motor proteins needed for sequencing were ligated using NEBNext^® Quick Ligation Module (NEB). AMPure XP beads (Beckman Coulter) at a concentration of 1x, 1x and 0.5x, respectively, were used to purify DNA between the library preparation steps. Final libraries were loaded onto R9.4.1 flow cells and samples were sequenced using a single MinION Mk 1B. To keep the sequencing throughput consistent, six biological samples were always pooled together and sequenced for 24 h. LR-PCR amplicons were pooled together and sequenced for 6 h.

The MinION sequencing library was produced using the ONT 1D genomic DNA by following the ligation (SQK-LSK108) protocol. In brief, 1.4 µg of DNA was fragmented in a Covaris g-TUBE (Covaris Ltd., Brighton, United Kingdom) by centrifugation to produce fragments of 8 kb in length. Sheared DNA was repaired using NEBNext FFPE repair mix (New England Biolabs, Ipswich, MA). End-repair and dA-tailing were performed with the NEBNext Ultra II End Repair/dA-Tailing module. Adapter ligation used the NEB Blunt/TA Ligase Master Mix, and the library was purified using Agencourt AMPure XP beads (Beckman Coulter Inc., Brea, CA).

Genomic DNA was size selected (10–50 kb for both organisms and 20–80 kb cut-offs for A120 only) using a BluePippin (Sage Science, Beverly, MA, USA) and repaired depending upon the DNA quantity recovered using the NEBNext FFPE Repair Mix (New England Biolabs, Ipswich, MA, USA). Following end-repair and 3′-A-tailing with the NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB), sequencing adapters provided by ONT (ONT Ltd., UK) were ligated using Blunt/TA Ligase Master Mix (NEB). Each library was then mixed with the running buffer with “fuel mix” and the library loading bead, and loaded on MinION R9.4 SpotON Flow Cells. Two and three libraries were run for the A25 and A120 strains, respectively. Read event data were generated by the MinKNOW control software (successive versions 1.3.25, 1.3.30, then 1.4.3 have been used) and base-calling done with the Metrichor software version 2.43.1, then 2.45.3 (1D base-calling RNN for LSK108 workflow). The data generated (pores metrics, sequencing, and base-calling data) by the MinION software was stored and organized using a Hierarchical Data Format. FASTA reads were extracted from MinION Hierarchical Data Format files using poretools [66 (link)].

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.