Ion 540 kit ot2 user guide

The 100 mg of fresh plant tissue was used to extract genomic DNA using the Genomic Mini AX Plant Spin kit (A&A Biotechnology, Gdańsk, Poland). The quality and concentration of the extracted DNA were verified using gel electrophoresis and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, United States). The genomic libraries were constructed with protocol: Ion Xpress™ Plus gDNA Fragment Library Preparation, using Ion Xpress Plus Fragment Library Kit (Pub. No. MAN0009847) (ThermoFisher Scientific, Waltham, MA, USA). Next-generation sequencing was performed on the GeneStudio™ S5 System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions using the protocol: Ion 540 ™ Kit—OT2 User Guide (Cat. No. A27753, Publication No. MAN0010850, Revision D). The reads generated from next-generation sequencing were assembled and annotated using the Geneious Prime 2020.2.5 package. Reads were mapped to MT735327 sequence from genbank using Genious mapper with default settings and minimum mapping quality 30%. Sequences that were mapped were subsequently assembled de novo using Geneious algorithm on default settings and annotated based on MT735327 sequence. The obtained 22 complete ITS2 sequences were submitted to GenBank. Each sequence was assigned an accession number (Table S2).

The genomic library was diluted to 100 pM. The concentration was measured on the Qubit™ 2.0 Fluorometer using Qubit™ dsDNA HS Assay Kit (Pub. No. MAN0002326 Revision: B.0) (Life Technologies). The P. uncinata template preparation was performed according to protocol: Ion PGM™ Hi-Q™ View OT2 Kit (Cat. No. A29900, Pub. No. MAN0014580 Rev. C.0). P. mugo and P. rotundata templates preparation were performed according to protocol: Ion 540™ Kit – OT2 (Cat. No A27753 Pub. No. MAN0010852 Rev. E.0). Evaluation of the templated Ion Sphere™ Particles (ISPs) was conducted using Ion Sphere™ Quality Control Kit (Cat.No. 4468656), according to protocol Ion Sphere™ Assay on the Qubit ™ 2.0 Fluorometer (Pub. No. MAN0016387 Revision A.0) (ThermoFisher Scientific, Waltham, MA, USA). P. uncinata genome sequencing was conducted on Ion 318™ Chip v2 BC by Ion Personal Genome Machine™ (PGM™) System (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s recommendations using protocol: Ion PGM™ Hi-Q™ View Sequencing Kit user guide (Cat. No. A30044, Pub. No. MAN0014583). Then, P. mugo and P. rotundata genome sequencing was conducted on Ion 540™ Chip by GeneStudio™ S5 System (Thermo Fisher Scientific, Waltham, USA) according to manufacturer’s recommendations using protocol: Ion 540™ Kit – OT2 User Guide (Cat. No A27753, Pub. No MAN0010850, Rev. D).