Sequel 2 device

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).

For long-read PacBio sequencing, high-molecular weight (HMW) DNA of C. japonica was isolated from root cultures using the NucleoBond HMW DNA kit (Macherey Nagel, Germany), quality was assessed with a FEMTOpulse device (Agilent, USA), and quantity was measured by the Quantus fluorometer (Promega, USA). A HiFi library was then prepared according to the “Procedure & Checklist - Preparing HiFi SMRTbell® Libraries using SMRTbell Express Template Prep Kit 2.0” manual with an initial DNA fragmentation by Megaruptor 3 (Diagenode, Belgium) and final library size binning into defined fractions by SageELF (Sage Science, USA). Size distribution was again controlled by FEMTOpulse (Agilent, USA). Polymerase-bound SMRTbell complexes were formed according to standard protocols (Pacific Biosciences of California Inc., USA) and loaded at an on-plate concentration of 85 pM (14, 15, 20, and 26 kb mean length). SMRT sequencing was performed using one 8 M SMRT cell per library (30 h movie time, 2 h pre-extension time) on the Pacific Biosciences Sequel II device, generating a total of 80 Gb (HiFi CCS). The SMRTbell libraries were sequenced at IPK Gatersleben.

DNA extraction, library preparation, and long-read sequencing of the NT1 and MN47 A. lyrata accessions were performed by the Max Planck-Genome-centre Cologne, Germany (https://mpgc.mpipz.mpg.de/home/). High molecular weight DNA was isolated from 1.5 g material with a NucleoBond HMW DNA kit (Macherey Nagel). Quality was assessed with a FEMTOpulse device (Agilent), and quantity was measured by a Quantus fluorometer (Promega). HiFi libraries were then prepared according to the manual “Procedure & Checklist—Preparing HiFi SMRTbell® Libraries using SMRTbell Express Template Prep Kit 2.0” with an initial DNA fragmentation by g-Tubes (Covaris) and final library size selection on BluePippin (Sage Science). Size distribution was again controlled by FEMTOpulse (Agilent). Size-selected libraries were then sequenced on a Sequel II device with Binding Kit 2.0 and Sequel II Sequencing Kit 2.0 for 30 h (Pacific Biosciences).