ERX10313171 webin-reads-FR01601_21.CCS_AmpliconSeq_ALS ERP133782 PRJEB49288 To generate ALS and ACCase amplicons for long-read PacBio sequencing we used the same DNA as for the ddRAD-seq libraries from the European collection. Before PCR amplification, DNA was normalized to 10 ng/μl. Then, 30 ng (ALS) and 50 ng (ACCase) total input DNA was used for the PCR Master Mix reaction (1 μl P5 indexing primer (5 μM), 1 μl P7 indexing primer (5 μM), 4 μl of 5x Prime STAR buffer, 1.6 μl dNTPs, 0.4 μl Prime STAR polymerase (Takara, R050B), filled up to 20 μl with water). The indexing PCR program for ALS was a 2-step PCR with 10 seconds of denaturation at 98°C and 210 seconds of annealing and extension at 68°C for 28 cycles, followed by a final extension for 10 min at 72°C. For ACCase, the annealing and extension step was elongated to 660 seconds. Amplicons were then pooled equally per gene and bead-cleaned. In the case of the 13.2 kb amplicon from ACCase, we added a BluePippin (SageScience) size selection to remove any remaining fragments below 10 kb. PacBio libraries were created according to the following PacBio amplicon protocol (part number 101-791-800 version 02 (April 2020)) and SMRT cells were loaded on a PacBio Sequel system with Binding Kit and Internal Ctrl Kit 3.0 (part number 101-461-600 version 10; October 2019). An extended hands-on protocol can be found online: https://github.com/SonjaKersten/Herbicide_resistance_evolution_in_blackgrass_2022 ERS8755982 SAMEA11106344 SMRTbell Express Template Prep Kit 2.0 AMPLICON GENOMIC PCR Sequel