Illumina HiSeq 2500 paired end sequencing; Nicotiana benthamiana
Genomic DNA was quantified using the Qubit dsDNA HS (High Sensitivity) Assay Kit (Q32854, Thermo Fisher Scientific).  The integrity of the DNA was checked using the FEMTO Pulse System Genomic DNA 165 kb Kit (FP-1002-0275, Agilent Technologies) which determined that 67% of the material was greater than 50kb in size.  The Chromium Controller instrument (10x Genomics) was used to produce a barcoded linked read library, with 1.25ng DNA input, using the Chromium™ Genome Library Kit & Gel bead Kit v2 (120258) following the Chromium Genome Reagent Kits Version 2 User Guide (CG00043).  The library yield was quantified using the Qubit dsDNA HS Assay, insert size was determined with Agilent 2100 Bioanalyzer High Sensitivity DNA chip (5067-4627, Agilent Technologies), and finally checked by qPCR (07960204001, Roche Diagnostics Ltd.) prior to sequencing.  The library was diluted to 2 nM with NaOH and 10μL transferred into 990μL HT1 (Illumina) to give a concentration of 20pM. The denatured library pool was further diluted in HT1 to an appropriate loading concentration in a volume of 420 μL, and spiked with 1% PhiX Control v3. This was then loaded on the Illumina HiSeq 2500 for on board cluster generation using a HiSeq Rapid SBS Kit v2 with HiSeq Control Software 2.2.68 and RTA 1.18.64. Sequencing data was demultiplexed and converted from base call (BCL) files to FASTQ files using the Illumina bcl2fastq2 conversion software (bcl2fastq version. 2.20.0), allowing for a one base-pair mismatch to the index sequence