Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes

• Published in: Biomedica : revista del Instituto Nacional de Salud Vol. 41; no. 4; pp. 773 - 786
• Main Authors: Gouvêa Moreira, Tiago César, Da Silva Spínola, Pricila, Campos Rezende, Micheline, Moreira de Freitas, Carla Simone, Borges Mury, Fábio, Rodrigues Bonvicino, Cibele, De Andrade Agostinho, Luciana
• Format: Journal Article
• Language: English
• Published: Colombia Instituto Nacional de Salud 15-12-2021
• Subjects: dna; False Positive Reactions; Genes, BRCA1; Genes, BRCA2; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Mutation; Original; Polymorphism, Single Nucleotide; Sensitivity and Specificity; sequence analysis; Sequence Analysis, DNA; high-throughput nucleotide sequencing; BRCA1; BRCA2; genes; Sequence analysis; DNA
• ISSN: 0120-4157; 2590-7379
• DOI: 10.7705/biomedica.5663

Next Generation Sequencing (NGS) is cost-effective and a faster method to study genes, but its protocol is challenging.
To analyze different adjustments to the protocol for screening the BRCA genes using Ion Torrent PGM sequencing and correlate the results with the number of false positive (FP) variants.
We conducted a library preparation process and analyzed the number of FP InDels, the library concentration, the number of cycles in the target amplification step, the purity of the nucleic acid, the input, and the number of samples/Ion 314 chips in association with the results obtained by NGS.
We carried out 51 reactions and nine adjustments of protocols and observed eight FP InDels in homopolymer regions. No FP Single-Nucleotide Polymorphism variant was observed; 67.5% of protocol variables were jointly associated with the quality of the results obtained (p<0.05). The number of FP InDels decreased when the quality of results increased.
The Ion AmpliSeq BRCA1/BRCA2 Community Panel had a better performance using four samples per Ion-314 chip instead of eight and the optimum number of cycles in the amplification step, even when using high-quality DNA, was 23. We observed better results with the manual equalization process and not using the Ion Library Equalizer kit. These adjustments provided a higher coverage of the variants and fewer artifacts (6.7-fold). Laboratories must perform internal validation because FP InDel variants can vary according to the quality of results while the NGS assay should be validated with Sanger.