임상검체에서 BRCA 돌연변이를 검출하는 차세대염기서열분석법의 임상적 효용성 평가
Evaluation of a Targeted Next-generation Sequencing Assay for BRCA Mutation Screening in Clinical Samples
서울대학교병원 진단검사의학과1, 엔젠바이오 연구개발센터2
Department of Laboratory Medicine1, Seoul National University Hospital, Seoul; Research & Development Centre2, NGeneBio Co., Ltd., Seoul, KoreaCorrespondence to:
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2021; 11(4): 283-289
Published October 1, 2021
Copyright © The Korean Society for Laboratory Medicine.
방법: 212명의 유방암 환자의 전혈에서 추출한 DNA 검체로부터 차세대염기서열분석법을 이용해 BRCA아큐테스트가 시행되었다. 분석대상은 단일염기서열변이(single nucleotide variation)와 짧은 삽입/결실(short insertion/deletion)이며 BRCA아큐테스트 결과를 전통적인 Sanger sequencing 결과와 비교하여 일치도를 평가하였다.
결과: 모든 타겟 영역은 평균 리드뎁스 20 이상으로 성공적으로 염기서열분석되었다. 단일염기서열변이와 짧은 삽입/결실 모두에서 Sanger sequencing 결과와 100% 일치하였다.
결론: 본 연구에서 BRCA아큐테스트는 우수한 분석능을 보여주었다. BRCA아큐테스트는 BRCA1/2 돌연변이 검사로 임상에서 유용하게 사용될 것으로 예상된다.
Methods: BRCAaccuTestTM is a reagent used to produce libraries for analyzing BRCA1/2 genes using next-generation sequencing (NGS), which analyzes blood-derived genomic DNA. Libraries with adapters and barcodes compatible with the Illumina platform were produced. The clinical performance of NGS-based BRCAaccuTestTM in identifying BRCA1/2 mutations was compared with that of the traditional Sanger sequencing method. Both NGS and Sanger sequencing were performed in a single laboratory using archival DNA from blood samples of 212 patients with breast cancer.
Results: All target regions amplified were successfully sequenced to obtain a minimum coverage of 20, demonstrating 100% concordance with the pathogenic single-nucleotide variations and small insertions-deletions previously identified by Sanger sequencing.
Conclusions: This study demonstrates the feasibility of using BRCAaccuTestTM to detect the BRCA1/2 mutations with high accuracy.
Breast and ovarian cancers exert substantial social and financial burdens. Breast cancer was one of the most commonly diagnosed cancers in 2018 in the United States . Every year, approximately 20,000 new cases of breast and ovarian cancers are detected in South Korea [2, 3]. The genetically well-characterized risk factors of breast and/or ovarian cancers include germline mutations of
The Sanger sequencing method is mainly used to detect mutations in
We aimed to evaluate the clinical performance of BRCAaccuTest™ (NGeneBio, Seoul, Korea). We evaluated the analytical performance of the BRCAaccuTest™ kit as an NGS-based in vitro diagnostic (IVD) reagent. We also evaluated the ability of an NGS analytic software called NGeneAnalySys™ (NGeneBio) to automatically detect mutations in
MATERIALS AND METHODS
1. Analytical validation of BRCAaccuTest™
To investigate the analytical performance of BRCAaccuTest™, control DNA samples were purchased from Coriell Institute (https://www.coriell.org/; See Supplemental Data Table S1). To test sensitivity (limit of detection), the input DNA was used at 50-, 10-, 5-, 1-, and 0.5-ng concentrations. Three reference DNAs, NA13713, NA14636, and NA14624, were used as positive controls of deleterious variants of either
2. Sample selection for clinical test
The clinical samples used in this study were obtained from the Seoul National University Hospital (SNUH). Briefiy, gDNA was extracted from patients who underwent
The residual gDNAs were maintained and controlled as per the regulations of the Ministry of Food and Drug Safety (MFDS) in the Republic of Korea and the Institutional Review Board (IRB) at SNUH. The samples satisfied the following criteria: 1) quantity was more than 100 ng for NGS or 5 μg for the Sanger method, 2) quality (A260/280) was 1.8–2.0, and 3) the storage period was less than 10 years. Samples were excluded from the study if they did not meet these criteria.
3. Clinical study design
This was a retrospective study performed by a single laboratory at SNUH under the regulation of MFDS in the Republic of Korea and IRB. It was a comparative study to assess the clinical utility and diagnostic consistency of BRCAaccuTest™ in comparison to the traditional Sanger sequencing method. As shown in Fig. 1, the target number of test samples was calculated based on the following criteria: diagnostic positive/negative agreement, 0.99; power, 80%; and statistical significance, 5%  with an additional 10% to compensate for drop-outs such as samples out of criteria. Therefore, the target number was initially set to 190 plus 22 extras for testing both
Figure 1. Design of the clinical study. The target number of test samples was determined according to the criteria of 0.99 diagnostic positive/negative agreement, 80% power, and 5% statistical significance. Abbreviations: PPA, positive percent agreement; NPA, negative percent agreement; OA, overall agreement.
4. NGS library preparation and sequencing
BRCAaccuTest™ was used to generate the NGS library of
5. Bioinformatic pipeline
The pipeline workfiow of data analysis was as follows: data QC, adapter trimming, alignment, variant calling, and annotation. Sequence reads generated were trimmed using Sickle v1.33 , a windowed adaptive trimming tool for sequencing adapters, and then aligned to the human hg19 reference genome using the BWA-MEM algorithm v0.7.10 . Genetic variants, including SNVs and short insertions/deletions (indels), were identified using GATK v2.3  and FreeBayes v9.9.2 , and the identified variants were annotated by snpEff v4.2 . This bioinformatic pipeline was fully automated in NGeneAnalySys™ software. Sequence reads generated as FASTQ files could be uploaded on NGeneAnalySys™ to analyze, annotate, classify, and visualize NGS sequencing results, including clinical reports. Variants were classified according to the ACMG Standards and Guidelines for the Interpretation of Sequence Variants . According to NGeneAnalySys™, the QC criteria for uniformity were set as follows: minimum coverage at >20× and average coverage at >200×.
6. Sanger sequencing
Peripheral blood samples were collected from family members of HBOC patients, non-familial breast cancer patients, or hereditary breast cancer patients. Genomic DNAs were extracted using the QIAGEN QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. The residual genomic DNAs were stored at -70°C after Sanger sequencing for
7. Statistical analysis
To investigate the precision of BRCAaccuTest™, its reproducibility was tested by changing experiment performers, laboratories, dates, and lot numbers. The experiments for all combinations were carried out with three positive controls (NA13713, NA14636, and NA14624) and one negative control (NA12878). All samples were duplicated.
1. Analytic performance of BRCAaccuTest™
The analytical performance of BRCAaccuTest™ was tested to confirm its sensitivity, specificity, and precision. The test results demonstrated the successful production of all the sequenced libraries with an average 300× coverage depth in the target regions. Each representative
2. Library preparation using BRCAaccuTest™
Approximately 10% of 212 test subjects (22 samples) were sequenced by the Sanger method to verify the stability of residual gDNA samples. All Sanger resequencing results were consistent with the previous ones. As the stability of the remaining samples was verified, subsequent NGS analysis was performed on 212 samples. In total, 207 samples (B001 to B207) were selected to create NGS libraries. These were divided into nine groups (runs) of 23 samples that were run on MiSeqDx® because the appropriate capacity of BRCAaccuTest™ is up to 24 samples, including 1 control (NA12878). The input DNA range of the 207 samples was 11.6–48.8 ng, and the average input DNA amount was 30.1±7.5 ng. The average time for library preparation for each group was 4.5–5.0 hours.
In total, 206 libraries of 207 samples were qualified and quantified at a success rate of 99.5%. Only one library, B021, was excluded owing to an insufficient amount of sample. As shown in Fig. 2A, all 206 libraries ranged from 15.2 to 100.3 nM, with an average of 55.2±15.0 nM (mean±SD). The BRCAaccuTest™ yielded 67.3 and 69.4 nM libraries with the input DNA of B15 (48.8 ng) and B101 (11.6 ng), respectively, indicating its highly consistent performance that was independent of input DNA amount. The average size of the final libraries, including the adapters, was 394.0±9.3 bp (Fig. 2B).
Figure 2. Quantity and size of the final libraries. (A) The libraries of samples B001 to B207 were quantified and represented in nanomole (nM) quantities in correlation to the amount of input DNA (ng). The black dashed lines indicate the range of the input DNA used in this study. The red dashed arrow represents the average concentration of the library. The blue dashed arrow represents the minimum concentration of the library. (B) The size of each library was individually determined and represented as a dot. The red dashed line indicates the average size (bp) of the library. The blue dashed lines represent the range of the library size (300–500 bp). B021 was excluded owing to an insufficient amount of sample.
We successfully performed nine runs of NGS. Overall QC rejection rate was 4%. Only one sample, B022, showed a 13× minimum coverage at a QC failure rate of 0.04%. Therefore, there was no issue in analyzing the variants, as the minimum coverage region was irrelevant (data not shown). In addition, 95.6% of samples met the QC criteria of the average coverage.
4. Variant analysis
For variant analysis, ubiquitous homozygous SNPs in Asian populations, including c.4563A>G (rs206075), c.6513G>C (rs206076), and c.7397T>C (rs169547), found in
The details of the most frequent variants predicted to be pathogenic or likely pathogenic using NGeneAnalySys™ are listed in Table 2. The pathogenic variant c.5496_5506delGGTGACCCGAGinsA (p.Val1833Serfs) in
5. Comparison with Sanger sequencing assay
The BRCAaccuTest™ results at all 199 positions were compared with those of the Sanger sequencing assay. Within the 198 individual samples, 1,640 mutations and 37,762 wild-type calls were detected. The agreement analysis results are shown in Table 3. Variant-level concordance (PPA and NPA) was 100% for all results with 95% confidence intervals of 99.8–100.0% for mutations (PPA) and 99.9–100.0% for wild-type location (NPA).
Many diagnostic laboratories use NGS technology to enhance throughput and reduce turn-around time and cost. However, NGS may introduce complexity resulting from the selection of components of the
The report described an NGS-based IVD reagent and analysis software for
NGS exhibits great potential by allowing rapid mutational analysis of multiple genes in HBOC . However, a reliable tool is desirable to take advantage of these opportunities to extract information from the big data generated by NGS. False-positive SNVs were directly adjacent to the 3′-end of one of the first PCR primers and were detected in only one strand [19, 20]. False-negative results have also been reported, missing the pathogenic
The bioinformatic pipelines provide an automated workfiow for the processing of NGS data for
This study has a limitation stemming from the exclusion of large genomic rearrangement in the NGS assay for
In conclusion, BRCAaccuTest™ can be exploited for clinical purposes, as it provided positive, negative, and overall diagnostic consistency of 100% with Sanger sequencing at a significance level of 5%. In addition, NGeneAnalySys™ can be used clinically to analyze data generated from BRCAaccuTest™ for screening pathogenic variants of the
Conflicts of Interest
Table S1. Control DNAs and their representative heterozygous mutations used in the analytical validation of BRCAaccuTest™lmo-11-4-283-supple1.pdf
Table S2. Interfering substances used in the specificity test of BRCAaccuTest™lmo-11-4-283-supple2.pdf
Table S3. Summary of 199 BRCA variants found in the clinical studylmo-11-4-283-supple3.pdf
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