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

Tiago César Gouvêa Moreira , Pricila da Silva Spínola , Micheline Campos Rezende, Carla Simone Moreira de Freitas , Fábio Borges Mury , Cibele Rodrigues Bonvicino, Luciana de Andrade Agostinho , .

DOI: https://doi.org/10.7705/biomedica.5663
Keywords: Sequence analysis, DNA, high-throughput nucleotide sequencing, genes, BRCA1, genes, BRCA2
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Abstract

Introduction: Next Generation Sequencing (NGS) is cost-effective and a faster method to study genes, but its protocol is challenging.
Objective: 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.
Material and methods: 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.
Results: 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.
Conclusion: 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.

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  • Tiago César Gouvêa Moreira Hospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Centro Universitário UNIFAMINAS, Muriaé, Brazil https://orcid.org/0000-0003-3362-7486
  • Pricila da Silva Spínola Divisão de Genética, Instituto Nacional do Câncer, Rio de Janeiro, Brazil; Programa de Pós-Graduação em Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil https://orcid.org/0000-0001-9121-3551
  • Micheline Campos Rezende Hospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Instituto de Ensino e Pesquisa Santa Casa BH, Belo Horizonte, Brazil https://orcid.org/0000-0001-6347-0040
  • Carla Simone Moreira de Freitas Hospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Instituto de Ensino e Pesquisa Santa Casa BH, Belo Horizonte, Brazil https://orcid.org/0000-0002-9397-9777
  • Fábio Borges Mury Thermo Fisher Scientific, São Paulo, Brazil https://orcid.org/0000-0002-8453-4354
  • Cibele Rodrigues Bonvicino Divisão de Genética, Instituto Nacional do Câncer, Rio de Janeiro, BrazilDivisão de Genética, Instituto Nacional do Câncer, Rio de Janeiro, Brazil; Programa de Pós-Graduação em Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil https://orcid.org/0000-0002-1948-7643
  • Luciana de Andrade Agostinho Hospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Centro Universitário UNIFAMINAS, Muriaé, Brazil; Programa de Pós-Graduação em Neurologia, Universidade Federal do Estado do Rio de Janeiro Rio de Janeiro, Brazil https://orcid.org/0000-0001-5604-8386

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How to Cite
1.
Gouvêa Moreira TC, da Silva Spínola P, Campos Rezende M, Moreira de Freitas CS, Borges Mury F, Rodrigues Bonvicino C, et al. Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes. Biomed. [Internet]. 2021 Dec. 15 [cited 2025 Apr. 4];41(4):773-86. Available from: https://revistabiomedicaorg.biteca.online/index.php/biomedica/article/view/5663

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Published
2021-12-15
Issue
Vol. 41 No. 4 (2021)
Section
Original articles

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