Ampliseq custom dna panel

Next-generation sequencing libraries were prepared from 112.5 ng genomic DNA with an AmpliSeq PLUS kit (Illumina, Singapore, Singapore), using an AmpliSeq Custom DNA Panel (Illumina, San Diego, CA, USA) for TP53 (Additional file 2, 3: Table S2-S3; exons only, 98.88% coverage, amplicons < 140 bp due to potential fragmentation from formalin-fixing, paraffin embedding process) as per the manufacturer’s protocol. Target amplification, using Illumina provided primers, was performed on a Veriti Thermal Cycler (Applied Biosystems, ThermoFisher Scientific, Socresby VIC, Australia). Amplicons were partially digested with the provided FuPa reagent (Illumina) before the index ligation reaction was carried out on a Veriti Thermal Cycler (Applied Biosystems). Libraries were cleaned up using AMPure XP Beads (Beckman Coulter, IN, USA), amplified on a Veriti Thermal Cycler and cleaned up again using AMPure XP Beads (Beckman Coulter) as per Illumina standard protocol. Libraries were checked for size on an Agilent Tapesation with Agilent D1000 Screen Tape and Reagents (Integrated Sciences, Chatswood NSW, Australia) and concentration was determined with the Qubit fluorometer using dsDNA High Sensitivity Assay kit (ThermoFisher Scientific, Socresby VIC, Australia) as per the manufacturer’s protocol. Two samples were excluded due to low library yields (< 2 nM).

The NGS [37 (link)] was run using an AmpliSeq Custom DNA panel (Illumina), which was designed using the Illumina DesignStudio Sequencing Assay Designer. The targeted NGS employed the amplicon sequencing method, where PCR primers were used to amplify the sequences of interest. Samples used for amplicon sequencing were transformed into libraries and enriched via PCR amplification individually and barcoded by ligating the indexes into the amplicons. The libraries were then pooled and analyzed using a bench-top sequencer. The panel in this study was designed to identify the 4 FHCGs, namely, LDLR, APOB-100, PCSK9, and LDLRAP1, and the HCAGs, namely, ABCG5 and ABCG8, with an overall gene coverage of 98.61% [38 (link)-40 (link)].

Genomic DNA was extracted from peripheral leukocytes using the phenol-chloroform method. The Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. For the HBB locus, all β-globin mutations and variants were confirmed by arrayed primer extension (APEX) or Sanger sequencing methods. The -158 (G→A) XmnI polymorphism in the HBG2 gene promoter (rs7482144) was confirmed by digestion of an amplified fragment. Genomic variations of NGS data were analyzed with Alamut Visual v.2.11 and v.2.13 and Illumina’s Variant Interpreter software. APEX and Sanger sequencing results were analyzed using Genorama PicDb Autoscan 7.0 software (Asper Biotech) and GenomeLab GeXP Genetic Analysis System v. 11.0 (Beckman Coulter), respectively. β^SHBB haplotypes were determined using a modification of the phase SNP method described by Shaikho et al. [11 (link)]. Detailed review of the full haplotype descriptions and analysis results with the newly described significant SNPs as well as β-thalassemia mutations detected in this study will be reported separately.