Sequel II sequencing; Raw reads: UK06481.poolA_100indiv.q30_HiFi_AmpliconSeq_ACCase.rep4
To generate the ACCase amplicons, we used a direct dual barcoding approach with target-specific primers that had the barcode sequences in their 5' ends. We conducted four independent PCR reactions (using different primer pairs) for each pool using 50 ng of DNA as a template. The 13.2 kb long target region was amplified using a master mix reaction with 1 μl Forward indexing primer (5 μM), 1 μl Reverse indexing primer (5 μM), 4 μl 5x Prime STAR buffer, 1.6 μl dNTPs (2.5 mM each), 0.4 μl Prime STAR GXL polymerase (1.25 U/μl) (Takara, R050B), filled up to 20 μl with water in a 2-step PCR reaction with 28 cycles (denaturation: 98°C, 10s; annealing: 68°C, 11 min; final extension: 72°C, 10 min; hold: 4°C). For a quality check, 5 µl of each amplicon pool was visualized on a 0.8% agarose gel and the concentration was determined with a Qubit™ system. Then, all amplicons were pooled equally into a large pool, bead-cleaned and size-selected using a BluePippin system (SageScience) with High-Pass Plus 0.75% agarose cassettes, 15 kb (342BPLUS03, Biozym). Only fragments larger than 10 kb were retained. The correct fragment size selection was verified with a Femto Pulse system (Agilent). The PacBio library was created following protocol no. 101-791-800 version 01 (June 2019) with the SMRTbell Express Template Prep Kit 2.0 (part number 100-938-900). Sequel II loading was performed according to manufacturer specifications with  Sequel II Binding Kit 2.0 and Int Ctrl 1.0 (part number 101-842-900). A detailed hands-on amplicon protocol can be found in Github: https://github.com/SonjaKersten/TSR_amplicon_pools. HiFi reads were generated with PacBio tools (https://github.com/PacificBiosciences/pbbioconda). This included the generation of circular consensus sequences (ccs) with ccs v6.0.0 with a minimum predicted accuracy of 0.999 (q30), demultiplexing of pools with lima v1.11.0 with parameter settings '--ccs --different --peek-guess --guess 80 --split-bam-named --min-ref-span 0.875 --min-scoring-regions 2', and conversion of the resulting bam to fastq format with bam2fastq v1.3.0.