Sqk lsk108

At the end of library preparation, each 1D barcoding sequencing adapter (BAM) (Oxford nanopore, EXP-NBD103) must be annealed to tethering oligonucleotides (tethers), which carry a hydrophobic group on their 5′end, included in the elution buffer (ELB) (Oxford nanopore, SQK-LSK108). This process is called tethering in the manufacturer’s protocol. Tethering in ELB buffer can assist the barcode sequencing adapters (BAM) to reach the nanopores faster. Motor proteins are pre-attached to BAM adapters. When a BAM adapter reaches a nanopore, the motor protein can unzip dsDNA into ssDNA and drive the resulting DNA strand through the nanopore at a fixed speed. The ideal tethering condition was determined by mixing the BAM adapter with ELB buffer in 1:1, 1:2, 1:3, and 1:4 ratios and incubated at 37° for 3 min. The BAM adapter was completely tethered at a 1:2 BAM: ELB ratio (Figure 2F). Tethering was also tested at 25° and on ice at a 1:2 ratio for 10 min but these conditions were less efficient than tethering at 37° for 3 min (data not shown).

The sequencing library was prepared following a modified one-direction (1D) Lambda control experiment protocol (Oxford Nanopore Technologies, SQK-LSK108). The ligation-based sequencing kit advises shearing 1 μg of Lambda genomic DNA to 8 kb fragments and spiking the sheared gDNA with a 3.6 kb positive control (Lambda genome 3'-end amplicon) in order to distinguish library preparation from sequencing failures. However, after validating our library preparation proficiency, we substituted 2 pg of B. subtilis spore DNA purified with Purelyse in lieu of the 3.6 kb positive control and replicated the Lambda control protocol without additional modifications. This substitution results in a low-input carrier library with ideal stoichiometry (Mojarro et al.,2018 (link)).

The amplicons were prepped using a Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies) with Native Barcoding Expansion (EXP-NBD103; ONT), according to the manufacturer’s protocol for 1D Native barcoding gDNA. The prepared library (12 µL at ~158 ng) was then combined with 35 µL of running buffer containing Fuel Mix (RBF) and 25.5 µL Library Loading beads (LLB, ONT) and loaded into a R9.4 flowcell (ONT) via the SpotON port according to the manufacturer’s instructions.

To obtain high molecular weight genomic DNA for long-read sequencing, we performed a DNA extraction from 750 μL the fibroblast cells using the Qiagen Blood and Cell Culture Mini Kit, following manufacturer’s instructions until the spooling step. We concentrated the DNA via centrifugation via EtOH precipitation, and then resuspended the pellet in 50 μL of TE buffer on a shaker at 22 °C overnight. We quantified DNA with a Qubit 2.0 dsDNA HS kit, and verified the DNA size distribution with a pulse-field gel using a 0.75% agarose TAE gel, run at 75V for 16 h, with the preset 5–150 kb program on the Pippin Pulse power supply, and estimated the DNA fragments to average roughly 23 kb in length.
We performed a ligation sequencing run (Oxford Nanopore, SQK-LSK108). For the Rapid library preparation, we used an input of 1000 ng of DNA, as recommended by the manufacturer. The DNA was end-repaired and ligated to sequencing adapters using the SQK-LSK108 kit according to manufacturer’s instructions. We quantified the sequencing libraries using a Qubit prior to sequencing. The sequencing library was run on a MinION instrument with an R9 flow cell using the NC 48 h sequencing protocol. We base-called fast5 raw reads generated from the sequencing run using Albacore v2.0.2 (Oxford Nanopore proprietary).

The target region was PCR amplified (primers 6L and 1R, ALLinRPH polymerase, 30 cycles). The resulting PCR product was checked by agarose electrophoresis, purified, and quantified on a Qubit fluorometer (Thermo Fisher Scientific, USA) according to standard procedures. The sequencing library was prepared using a 1D Ligation Sequencing kit (SQK-LSK108, Oxford Nanopore Technologies, UK). The library was run on a MinION device using the FLO-MIN107 R9 Flow Cell, according to the manufacturer´s instructions (Oxford Nanopore Technologies, UK). Base-calling was performed using ONT Albacore software v.2.1.10. The reads were assembled using Canu software v.1.7.1^{21 (link)}.

Genomic DNA (5 µg) was sheared to ~5–25-kilobase fragments using Megaruptor^® 2 (Diagenode, B06010002) and was then size-selected (10–30 kilobases) with a BluePippin device (Sage Science, MA) to remove the small DNA fragments. Subsequently, genomic libraries were prepared using the Ligation Sequencing 1D Kit (SQK-LSK108, Oxford Nanopore, UK). End-repair and dA-tailing of DNA fragments were performed using the Ultra II End Prep module (New England Biolabs, E7546L), according to manufacturer’s recommended protocols. Finally, the purified dA-tailed sample was incubated with blunt/TA ligase master mix (#M0367, NEB), tethered with 1D adapter mix from the SQK-LSK108 Kit (Oxford Nanopore Technologies), and purified. The resulting library was sequenced on R9.4 flow cells using GridION X5.

The Oxford Nanopore Technologies (Oxford, United Kingdom) MinION sequencer was used to obtain long reads to span repetitive elements and close genomes and plasmids. DNA extraction was performed using the Genomic-tip 100/G kit (Qiagen, Hilden, Germany). Library preparation was carried out according to manufacturer’s indications using a combination of Native Barcoding Kit 1D and Ligation Sequencing Kit 1D; EXP-NBD103 and SQK-LSK108 (Oxford Nanopore Technologies, Oxford, United Kingdom), respectively.
The tool Albacore (Oxford Nanopore Technologies, Oxford, United Kingdom) was used for demultiplexing the reads which were later used to perform the Canu assembly (Koren et al., 2017 (link)). A hybrid assembly combining previous MiSeq short reads with MinION-generated long reads was performed using a hybridSpades (Antipov et al., 2016 (link)).