Ion onetouch 2

An exome amplicon library was prepared using the Ion AmpliSeq Exome RDY kit (Thermo Fisher, Waltham, MA, USA). Briefly, 100 ng of genomic DNA was added to dehydrated, ultra-high multiplexed primer pairs (12 pools) in a 96-well plate and amplified with the following PCR conditions: 99 °C for 2 min; 99 °C for 15 s and 60 °C for 16 min (10 cycles); and holding at 10 °C. Primers were partially digested using a FuPa reagent, and then sequencing motifs and barcodes were ligated to the amplicons. The library was purified using the Agencourt AMPure XP Reagent (Beckmann Coulter, Brea, CA, USA). The concentration of the final library was determined using the Ion Library TaqMan Quantitation Kit (Thermo Fisher, Waltham, MA, USA) on an ABI 7500 qPCR instrument with the absolute quantification method.
Template preparation was performed with the Ion 540 OT2 Kit (Thermo Fisher, Waltham, MA, USA) on semi-automated Ion OneTouch 2 instrument using the emPCR method. After breaking the emulsion, non-templated beads were removed from the solution during the enrichment process on the Ion OneTouch ES (Thermo Fisher, Waltham, MA, USA) machine. Subsequently, the sequencing primer and polymerase were added, the fully prepared Ion sphere particles were loaded into an Ion 540 chip, and sequencing runs were performed using the Ion S5 Sequencing kit (Thermo Fisher, Waltham, MA, USA) with 500 flows.

Library amplification was performed in the Veriti^® Thermal Cycler (Applied Biosystems) using reagents from the HID Ion AmpliSeq™ Identity Panel kit²⁴ . The amplification reaction contained 5X Ion AmpliSeq™ HiFi Master Mix (4 ul) and 2X HID-Ion AmpliSeq™ Identity SNP-124 Panel (10 ul). PCR cycling was reduced to 18 cycles for FTA™ discs. Partial digestion of primers was performed by treating the amplicons with FuPa Reagent (2 ul) (Thermo Fisher Scientific). This was followed by the ligation of barcodes to the amplicons using Switch Solution (4 ul), DNA ligase (2 ul), and Ion Xpress Barcode (2 ul) (Thermo Fisher Scientific). Barcoded libraries were purified with Agencourt AMPure XP Reagents (Beckman Coulter, Brea, CA) and quantified using the Ion Library TaqMan PCR Mix (Thermo Fisher Scientific) and the 20X Quantitation Assay (Thermo Fisher Scientific). Pooling of the library was performed by mixing equal volumes of barcoded samples with a concentration of 20 pM. Clonal amplification via emulsion PCR and library enrichment was performed using the Ion OneTouch™ 2 (OT2) System with the Ion PGM™ Template OT2 200 Kit and the Ion OneTouch™ ES (Thermo Fisher Scientific). Sequencing was performed in the Ion Torrent PGM™ (Thermo Fisher Scientific) with Ion PGM™ Sequencing 200 Kit and the Ion 318 Chip Kit v2 (Thermo Fisher Scientific) following manufacturer’s protocol.

The libraries were prepared using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific), following the manufacturer’s recommendations [41 ]. All the libraries were evaluated for their quality on the Agilent 2100 Bioanalyzer instrument. Subsequently, the libraries were pooled in an equimolar amount into the template reaction for attachment of the fragments to Ion Sphere Particles (ISP) and clonal amplification in emulsion-PCR. The template reaction was conducted on the Ion OneTouch 2 instrument (Thermo Fisher). Next, recovery and enrichment were performed. Enriched samples were subsequently sequenced on the Ion GeneStudio S5 System (Thermo Fisher), using the Ion 520 chip, which produced 3–5 million reads (1–2 Gb).