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

References

Niell BL, Rennert G, Bonner JD, Almog R, Tomsho LP, Gruber SB. BRCA1 and BRCA2 founder mutations and the risk of colorectal cancer. J Natl Cancer Inst. 2004;96:15-21. https://doi.org/10.1093/jnci/djh008

Garber JE, Syngal S. One less thing to worry about: The shrinking spectrum of tumors in BRCA founder mutation carriers. J Natl Cancer Inst. 2004;96:2-310. https://doi.org/1093/jnci/djh021

Wallace AJ. New challenges for BRCA testing: A view from the diagnostic laboratory. Eur J Hum Genet. 2016;24(Suppl.1):S10-18. https://doi.org/10.1038/ejhg.2016.94

Loeffelholz M, Fofanov Y. The main challenges that remain in applying high-throughput sequencing to clinical diagnostics. Expert Rev Mol Diagn. 2015;15:1405-8. https://doi.org/10.1586/14737159.2015.1088385

Germani A, Libi F, Maggi S, Stanzani G, Lombardi A, Pellegrini P, et al. Rapid detection of copy number variations and point mutations in BRCA1/2 genes using a single workflow by ion semiconductor sequencing pipeline. Oncotarget. 2018;9:33648-55. https://doi.org/10.18632/oncotarget.26000

Zanella I, Merola F, Biasiotto G, Archetti S, Spinelli E, Di Lorenzo D. Evaluation of the Ion Torrent PGM sequencing workflow for the routine rapid detection of BRCA1 and BRCA2 germline mutations. Exp Mol Pathol. 2017;102:314-20. https://doi.org/10.1016/j.yexmp.2017.03.001

Oosterwijk JC, de Vries J, Mourits MJ, de Bock GH. Genetic testing and familial implications in breast-ovarian cancer families. Maturitas. 2014;78:252-7. https://doi.org/10.1016/j.maturitas.2014.05.002

Claus EB, Schildkraut JM, Thompson D, Risch NJ.The genetic attributable risk of breast and ovarian cancer. Cancer. 1996;77:2318-24. https://doi.org/10.1002/(SICI)1097-0142(19960601)77:11<2318::AID-CNCR21>3.0.CO;2-Z

Lih CJ, Harrington RD, Sims DJ, Harper KN, Bouk CH, Datta V, et al. Analytical validation of the next-generation sequencing assay for a nationwide signal-finding clinical trial: Molecular analysis for therapy choice clinical trial. J Mol Diagn. 2017;19:313-27. https://doi.org/10.1016/j.jmoldx.2016.10.007

Maistro S, Teixeira N, Encinas G, Katayama ML, Niewiadonski VD, Cabral LG, et al. Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil. BMC Cancer. 2016;16:934. https://doi.org/10.1186/s12885-016-2966-x

Bragg LM, Stone G, Butler MK, Hugenholtz P, Tyson GW. Shining a light on dark sequencing: Characterising errors in Ion Torrent PGM data. PLoS Comput Biol. 2013;9:e1003031. https://doi.org/10.1371/journal.pcbi.1003031

Kang HP, Maguire JR, Chu CS, Haque IS, Lai H, Mar-Heyming R, et al. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing. Peer J. 2016;4:e2162. https://doi.org/10.7717/peerj.2162

Park J, Jang W, Chae H, Kim Y, Chi HY, Kim M. Comparison of targeted next-generation and Sanger sequencing for the BRCA1 and BRCA2 mutation screening. Ann Lab Med. 2016;36:197-201. https://doi.org/201603197

Mehta A, Vasudevan S, Sharma SK, Kumar D, Panigrahi M, Suryavanshi M, et al. Germline BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance associated with breast/ovarian cancer: A report from North India. Cancer Manag Res. 2018;10:6505-16. https://doi.org/10.2147/CMAR.S186563

Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med. 2013;15:733-47. https://doi.org/10.1038/gim.2013.92

Trujillano D, Weiss ME, Schneider J, Koster J, Papachristos EB, Saviouk V, et al. Nextgeneration sequencing of the BRCA1 and BRCA2 genes for the genetic diagnostics of hereditary breast and/or ovarian cancer. J Mol Diagn. 2015;17:162-70. https://doi.org/10.1016/j.jmoldx.2014.11.004

Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463-7. https://doi.org/10.1073/pnas.74.12.5463

Vendrell JA, Vilquin P, Larrieux M, van Goethem C, Solassol J. Benchmarking of ampliconbased next-generation sequencing panels combined with bioinformatics solutions for germline BRCA1 and BRCA2 alteration detection. J Mol Diagn. 2018;20:754-64. https://doi.org/S1525-1578(18)30072-2 19

Jennings LJ, Arcila ME, Corless C, Kamel-Reid S, Lubin IM, Pfeifer J, et al. Guidelines for validation of next-generation sequencing-based oncology panels: A joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19:341-65. https://doi.org/S1525-1578(17)30025-9

Beck TF, Mullikin JC, Program NCS, Biesecker LG. Systematic evaluation of Sanger validation of next-generation sequencing variants. Clin Chem. 2016;62:647-54. https://doi.org/10.1373/clinchem.2015.249623

Buzolin AL, Moreira CM, Sacramento PR, Oku AY, Fornari A, Antonio DSM, et al. Development and validation of a variant detection workflow for BRCA1 and BRCA2 genes and its clinical application based on the Ion Torrent technology. Hum Genomics. 2017;11:14. https://doi.org/10.1186/s40246-017-0110-x

Harismendy O, Ng PC, Strausberg RL, Wang X, Stockwell TB, Beeson KY, et al. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol. 2009;10:R32. https://doi.org/10.1186/gb-2009-10-3-r32

Strom CM, Rivera S, Elzinga C, Angeloni T, Rosenthal SH, Goos-Root D, et al. Development and validation of a next-generation sequencing assay for BRCA1 and BRCA2 variants for the clinical laboratory. PLoS ONE. 2015;10:e0136419. https://doi.org/10.1371/journal.pone.0136419

Bijwaard K, Dickey JS, Kelm K, Tezak Z. The first FDA marketing authorizations of next generation sequencing technology and tests: Challenges, solutions and impact for future assays. Expert Rev Mol Diagn. 2015;15:33-40. https://doi.org/10.1586/14737159.2015.979795

Gargis AS, Kalman L, Berry MW, Bick DP, Dimmock DP, Hambuch T, et al. Assuring the quality of next-generation sequencing in clinical laboratory practice. Nat Biotechnol. 2012;30:1033-6. https://doi.org/10.1038/nbt.2403

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. biomedica [Internet]. 2021 Dec. 15 [cited 2024 May 19];41(4):773-86. Available from: https://revistabiomedica.org/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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