Background The genetic variant p.R337H in the TP53 gene is a pathogenic variant associated with Li-Fraumeni Syndrome and an increased risk of developing various types of cancer, including breast cancer. This variant is present in about 1 in every 300 individuals in the Southern region of Brazil, likely due to a founder effect. Amplicon sequencing via the Sanger methodology remains prevalent for the genotyping of such variants. The spectrum of applications for methods aimed at identifying human genetic variants spans from the exploration of pathogenic mutations to pharmacogenetic studies. Nevertheless, the use of conventional NGS techniques, such as Illumina, for this application is still financially costly. Third-generation sequencing by nanopores has low implementation costs and has seen expanded use recently, particularly due to genomic surveillance studies conducted during the pandemic, and after the release of the Q20+ chemistry, it may be an interesting alternative for human variant research. Thus, the present study aims to validate a diagnostic test for researching this variant through nanopore sequencing, including the development of a simplified analysis pipeline. Methods 24 blood samples with previously known results underwent nucleic acid extraction using the Maxwell extractor (Promega) with the Maxwell RSC Blood DNA kit. The extraction eluate was used in a PCR reaction. The exon 10 region of the TP53 gene was amplified using specific primers. Amplification confirmation was performed by agarose gel electrophoresis. Each sample received a barcode through a ligation reaction using NEB Blunt/TA Ligase Master Mix (NEB) according to the manufacturer's instructions. The sequencing library was prepared using the SQK-NBD114.24 kit (Nanopore) and sequenced on a flongle R10.1.4 using the Flongle Sequencing Expansion kit (EXP-FSE002). Sequencing was configured for 4 hours in the MinION control software (MinKnow). Basecalling was performed using Guppy_barcoder in simplex and duplex modes using the super accuracy algorithm. Reads were demultiplexed using a pipeline adapted from RAMPART and mapped to the reference using the minimap2 assembler implemented in Geneious. A mapping and variant calling workflow was created in Geneious software for batch analysis. Results The 24 PCR reactions produced amplicons of the expected size. The Rampart tool was adapted for exon 10 of the TP53 gene and allowed real-time monitoring of coverage per sample. Approximately 90 thousand reads were generated in total, with an average of 3600 reads per sample. The analyses showed that simplex and duplex basecalling methods are effective for analyzing the presence of the variant in question, however, simplex generated greater coverage. Comparison with Sanger sequencing revealed an accuracy of 100%. Conclusions Our results show that genotyping of the p.R337H variant in the TP53 gene can be performed using nanopore sequencing with the new Q20+ chemistry. The coverage obtained for 24 samples on a flongle (low-cost flow cell) in a reduced sequencing time suggests that sequencing capacity using this methodology can be much higher. Additionally, the developed analysis pipeline simplifies the workflow and allows it to be performed without the requirement of a highly specialized bioinformatics professional.

中文翻译：

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B-194 通过纳米孔测序验证 TP53 基因中 p.R337H 变体的诊断测试：开发简化的分析流程


背景 TP53 基因中的遗传变异 p.R337H 是一种与 Li-Fraumeni 综合征相关的致病变异，并且会增加患各种类型癌症（包括乳腺癌）的风险。在巴西南部地区，大约每 300 个人中就有 1 人存在这种变异，这可能是由于创始人效应造成的。通过桑格方法进行的扩增子测序对于此类变异的基因分型仍然很普遍。旨在识别人类遗传变异的方法的应用范围涵盖从致病突变的探索到药物遗传学研究。然而，使用传统的 NGS 技术（例如 Illumina）进行此应用仍然需要高昂的成本。纳米孔第三代测序实施成本低，最近得到了广泛使用，特别是由于大流行期间进行的基因组监测研究，并且在 Q20+ 化学物质发布后，它可能是人类变异研究的一个有趣的替代方案。因此，本研究旨在验证通过纳米孔测序研究该变体的诊断测试，包括开发简化的分析流程。方法 使用 Maxwell 提取器 (Promega) 和 Maxwell RSC 血液 DNA 试剂盒对先前已知结果的 24 份血液样本进行核酸提取。提取洗脱液用于PCR反应。使用特异性引物扩增 TP53 基因的外显子 10 区域。通过琼脂糖凝胶电泳进行扩增确认。每个样品根据制造商的说明，使用 NEB Blunt/TA Ligase Master Mix (NEB) 通过连接反应获得条形码。使用SQK-NBD114制备测序文库。24 试剂盒 (Nanopore) 并使用 Flongle 测序扩展试剂盒 (EXP-FSE002) 在 flongle R10.1.4 上进行测序。在 MinION 控制软件 (MinKnow) 中将测序配置为 4 小时。使用 Guppy_barcoder 在单工和双工模式下使用超准确算法进行碱基识别。使用改编自 RAMPART 的管道对读取进行多路分解，并使用 Geneious 中实现的 minimap2 汇编器映射到参考。在 Geneious 软件中创建了映射和变体调用工作流程以进行批量分析。结果 24 个 PCR 反应产生了预期大小的扩增子。 Rampart 工具适用于 TP53 基因的外显子 10，并允许实时监控每个样本的覆盖范围。总共生成了大约 9 万个读数，每个样本平均有 3600 个读数。分析表明，单工和双工碱基检出方法对于分析相关变体的存在是有效的，然而，单工产生了更大的覆盖范围。与桑格测序的比较显示准确度为100%。结论 我们的结果表明，可以使用纳米孔测序和新的 Q20+ 化学方法对 TP53 基因中的 p.R337H 变体进行基因分型。在缩短的测序时间内，在 flongle（低成本流动池）上获得 24 个样本的覆盖率表明，使用这种方法的测序能力可以高得多。此外，开发的分析流程简化了工作流程，无需高度专业的生物信息学专业人员即可执行。