B-194 Validation of a Diagnostic Test for the p.R337H Variant in the TP53 Gene by Nanopore Sequencing: Development of a Simplified Analysis Pipeline

Abstract

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.