Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes
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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Instituto Nacional de Salud
2021-12-01
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| Series: | Biomédica: revista del Instituto Nacional de Salud |
| Subjects: | |
| Online Access: | https://revistabiomedica.org/index.php/biomedica/article/view/5663 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Tiago César Gouvêa Moreira |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T03:33:03Z |
| format | Article |
| id | doaj.art-8fdcb9a86d294d89bce01e47e60ba678 |
| institution | Directory Open Access Journal |
| issn | 0120-4157 |
| language | English |
| last_indexed | 2024-04-14T03:33:03Z |
| publishDate | 2021-12-01 |
| publisher | Instituto Nacional de Salud |
| record_format | Article |
| series | Biomédica: revista del Instituto Nacional de Salud |
| spelling | doaj.art-8fdcb9a86d294d89bce01e47e60ba6782022-12-22T02:14:52ZengInstituto Nacional de SaludBiomédica: revista del Instituto Nacional de Salud0120-41572021-12-0141477378610.7705/biomedica.56635663Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genesTiago César Gouvêa Moreira0Pricila da Silva Spínola1Micheline Campos Rezende2Carla Simone Moreira de Freitas3Fábio Borges Mury4Cibele Rodrigues Bonvicino5Luciana de Andrade Agostinho6Hospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Centro Universitário UNIFAMINAS, Muriaé, 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, BrazilHospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Instituto de Ensino e Pesquisa Santa Casa BH, Belo Horizonte, BrazilHospital do Câncer de Muriaé, Fundação Cristiano Varella, Muriaé, Brazil; Instituto de Ensino e Pesquisa Santa Casa BH, Belo Horizonte, BrazilThermo Fisher Scientific, São Paulo, BrazilDivisão 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, BrazilHospital 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, BrazilIntroduction: 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.https://revistabiomedica.org/index.php/biomedica/article/view/5663sequence analysisdnahigh-throughput nucleotide sequencinggenes, brca1genes, brca2 |
| spellingShingle | 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 Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes Biomédica: revista del Instituto Nacional de Salud sequence analysis dna high-throughput nucleotide sequencing genes, brca1 genes, brca2 |
| title | Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes |
| title_full | Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes |
| title_fullStr | Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes |
| title_full_unstemmed | Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes |
| title_short | Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes |
| title_sort | correlation between the number of false positive variants and the quality of results using ion torrent pgm™ sequencing to screen brca genes |
| topic | sequence analysis dna high-throughput nucleotide sequencing genes, brca1 genes, brca2 |
| url | https://revistabiomedica.org/index.php/biomedica/article/view/5663 |
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