An experience of clinical application of NGS sequencing technology on MiSeq illumina platform for BRCA1 and BRCA2 mutations diagnostics

N.A. Ermolenko; U.A. Boyarskykh; A.A. Kechin; A.F. Lazarev; V.D. Petrova; N.E. Kushlinskii; M.L. Philipenko

An experience of clinical application of NGS sequencing technology on MiSeq illumina platform for BRCA1 and BRCA2 mutations diagnostics

N.A. Ermolenko¹, U.A. Boyarskykh², A.A. Kechin³, A.F. Lazarev⁴, V.D. Petrova⁵, N.E. Kushlinskii⁶, M.L. Philipenko⁷

research article Pages: 11-23

¹ Junior Research Scientist, Laboratory of Pharmacogenomics, Institute of Chemical Biology and Fun-damental Medicine Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), Novosibirsk
² Ph.D. (Biol.), Junior Research Scientist, Laboratory of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), Novosibirsk
³ Post-graduate Student, Laboratory of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), Novosibirsk State University
⁴ Dr.Sc. (Med.), Professor, Altai branch N.N. Blokhin Russian Cancer Research Center
⁵ Ph.D. (Med.), Altai branch N.N. Blokhin Russian Cancer Research Center. A.M. Mazitova  Student, Kazan Federal University
⁶ Dr.Sc. (Med.), Professor, Head of the Clinical Biochemistry Laboratory, N.N. Blokhin Russian Cancer Research Center, Moscow
⁷ Ph.D. (Biol.), Head of the laboratory of pharmacogenomics, Institute of Chemical Biology and Funda-mental Medicine Siberian branch of the Russian Academy of Sciences, Novosibirsk

Keywords: NGS BRCA1 BRCA2 Illumina MiSeq hereditary breast cancer massively parallel sequencing

Abstract

The aim of this study was to evaluate the Illumina MiSeq massively parallel sequencing platform for routine clinical prac-tice. We implemented an amplicon-based method of the BRCA1 and BRCA2 genes resequencing to identify disease-causing mutations in hereditary breast cancer (HBC) patients. To analyze the results we designed our own workflow based on a combination of the freely available softwares. The study design included two parts. Firstly, we developed data analysis workflow by testing of a «training samples» (16 DNA samples with known BRCA1 and BRCA2 exons sequence). Secondly, the workflow was validated in a set of 80 samples from HBC patients. In order to determine variants, our workflow includes a set of filters: sequencing depth (>20%), variant allele frequency (>14%), FS (Phred-scased p-value using Fisher-s exact test to detect strand bias) and variant Quality (Q-score). As a result, 46 unique variants there were identified. Using the 1000 Genomes Project as a comparison group we determined nine nonsynonymous rare (allele frequency <0,001 in 1000 Genomes Project) mutations: (g. 41246297A→C, g. 41226488C→A, g. 41256236_41256237delGG, g. 32930673C→T, g. 32893369G→C, g. 32906593C→A, g. 32907129T→C, g. 32914151A→G, g. 32929242_32929243delAG; coordinates specified for GRCh37). We assume this variants may be disease-causing mutations in (HBC) patients g. 41246297A→C, g. 41226488C→A, g. 41256236_41256237delGG (gene BRCA1), g. 32930673C→T, g. 32893369G→C, g. 32906593C→A, g. 32907129T→C, g. 32914151A→G, g. 32929242_32929243delAG (gene BRCA2). The workflow developed in this study con-forms the sensitivity (98.6%) and specificity (94.4%) requirements for the clinical application and may be useful for genetic diagnosis of HBCS.

How to cite

Ермоленко Н.А., Боярских У.А., Кечин А.А., Лазарев А.Ф., Петрова В.Д., Мазитова А.М., Филипенко М.Л. Опыт клинического использования платформы MiSeq illumina для диагностики мутаций BRCA1 и BRCA2 // Технологии живых систем. 2015. № 1. С. 11–23.

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[2] Abecasis G.R., Auton A., Brooks L.D., et al.An integrated map of genetic variation from 1,092 human genomes // Nature. 2012. V. 491. № 7422. P. 56-65.

[3] Chan M., Ji S.M., Yeo Z.X., et al. Development of a next-generation sequencing method for BRCA mutation screening: a comparison between a high-throughput and a benchtop platform // J. Mol. Diagn. 2012. V. 14. № 6. P. 602-612.

[4] Choi Y., Sims G.E., Murphy S., et al. Predicting the functional effect of amino acid substitutions and indels // PLoS One. 2012. V. 7. № 10:e46688. doi:10.1371/journal.pone.0046688.

[5] De Leeneer K., Hellemans J., De Schrijver J., et al. Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations // Hum. Mutat. 2011. V. 32. № 3. P. 335-344.

[6] Diego S. Entire document and oligonucleotide sequences. Illumina, Inc. 2007-2013. 2014. P. 1-28.

[7] Feliubadaló L., Lopez-Doriga A., Castellsagué E., et al. Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes // Eur. J. Hum. Genet. 2013. V. 21. № 8. P. 864-870.

[8] Hernan I., Borràs E., de Sousa Dias M., et al. Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing // J. Mol. Diagn. 2012. V. 14. № 3. P. 286-293.

[9] Kumar P., Henikoff S., Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm // Nat. Protoc. 2009. V. 4. № 7:1073-1081. doi:10.1038/nprot.2009.86.

[10] Li H., Handsaker B., Wysoker A., et al.The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009. V. 25. № 16. P. 2078-2079.

[11] Li H., Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009. V. 25. № 14. P. 1754-1760.

[12] McKenna A., Hanna M., Banks E., et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data // Genome Res. 2010. V. 20. № 9. P. 1297-1303.

[13] Michils G., Hollants S., Dehaspe L., et al. Molecular analysis of the breast cancer genes BRCA1 and BRCA2 using amplicon-based massive parallel pyrosequencing // J. Mol. Diagn. 2012. V. 14. № 6. P. 623-630.

[14] Morgan J.E., Carr I.M., Sheridan E., et al. Genetic diagnosis of familial breast cancer using clonal sequencing // Hum. Mutat. 2010. V. 31. № 4. P. 484-491.

[15] Stratton M.R., Rahman N. The emerging landscape of breast cancer susceptibility // Nat. Genet. 2008. V. 40. № 1. P. 17-22.

[16] Thompson J.F., Reifenberger J.G., Giladi E., et al. Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations // Genome Res. 2012. V. 22. № 2. P. 340-345.

[17] Wang K., Li M., Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data // Nucleic Acids Res. 2010. V. 38. № 16:e164. doi:10.1093/nar/gkq603.