Shotgun and SMRT Sequencing of Infected Carrots

Shotgun sequencing of extracted DNA from infected carrot samples M8 and M33 (Figure 1) was performed separately for each sample using two sequencing technologies. To generate short reads with paired ends, Illumina sequencing [28 (link)] was carried out on the HiSeq 2500 platform (Illumina, San Diego, CA, USA). For long-read sequencing, single-molecule real-time sequencing (SMRT) sequencing [29 (link)] was performed on the Sequel IIe platform (Pacific Biosciences, Menlo Park, CA, USA). First, DNA was enriched for longer fragments through a 0.45% (v/v) PB AMPure bead purification step (Pacific Biosciences). Barcoded libraries were then prepared according to the manufacturer’s protocol “Preparing HiFi Libraries from Low DNA Input Using SMRTbell Express Template Prep Kit 2.0” (Pacific Biosciences). Libraries were equimolarly pooled and sequenced on a Sequel II device (Pacific Biosciences) using a Sequel II binding kit 2.0, Sequel II sequencing chemistry 2.0, and an 8M ZMW SMRT cell for 30 h (Pacific Biosciences). Sequencing data were demultiplexed and high-fidelity data were generated using the SMRTlink Suite v.9.0 (Pacific Biosciences) with default settings. The NGS approaches were conducted by the Max Planck Genome Centre Cologne (Cologne, Germany).