Unique dual index adapters

Libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit with the IDT for Illumina Unique Dual Index adapters following manufacturer’s recommendations. Completed libraries were QC’d and quantified using a combination of Qubit dsDNA HS and Agilent 4200 TapeStation High Sensitivity DNA 1000 assays. The libraries were pooled in equimolar amounts and the pool quantified using the Kapa Biosystems Illumina Library Quantification qPCR kit. This pool was loaded onto an Illumina NextSeq 500/550 High Output flow cell (v2.5) and sequencing performed in a 1x75 bp single read format using a NextSeq 500/550 High Output 75 cycle reagent kit (v2.5). Base calling was done by Illumina Real Time Analysis (RTA) v2.4.11 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1. Sequence data have been accessioned in NCBI’s SRA under the BioProject PRJNA704083.

Genomic DNA for Oxford Nanopore Technologies (ONT) sequencing was isolated from greenhouse grown leaves of DM1S1 as described previously (Vaillancourt and Buell 2019 (link)). Short sequences were removed using the Circulomics Short Read Eliminator Kit (Circulomics, Baltimore, MD, Cat #SS-100-101-01). Eleven ONT DNA libraries were prepared using the ONT SQK-LSK109 Ligation Sequencing kit and sequenced on six R9 ONT FLO-MIN106 Rev D flow cells. Five of these R9 ONT flow cells were washed and reused according to the Flow Cell Wash kit and protocol (EXP-WSH003, version: WFC_9088_v1_revB_18Sept2019). Sequencing was performed using default settings on an ONT (Oxford, UK) MinION (MinIon 19.12.5 or 19.10.1) using MinKNOW default settings (MinKNOW v3.5.5, v3.6.0, v3.6.5). Details of the sequence generation are provided in Supplementary Table 1.
Genomic DNA for whole-genome shotgun sequencing (WGS) was isolated from young leaves of tissue culture-grown DM1S1 and 1S1 clones using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Illumina TruSeq Nano DNA WGS libraries were prepared and multiplexed using IDT Illumina Unique Dual Index adapters, then sequenced on an Illumina HiSeq 4000 in paired-end mode by the Michigan State University Genomics Core, generating 150 nt reads (Supplementary Table 1).