José Padilla Ruiz¹, Belén de la Morena Barrio¹, Eugenia de la Morena Barrio¹, Pedro Garrido Rodríguez², ROSA CIFUENTES RIQUELME¹, Carlos BRAVO PEREZ¹, ANTONIA MIÑANO NAVARRO¹, Vicente Vicente García¹, Javier CORRAL DE LA CALLE¹

(1) HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL, IMIB-Arrixaca, España
(2) Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia, Spain., 30003, España (Región de Murcia)

Since Sanger developed his method to sequence DNA in 1977, sequencing methods have significantly evolved through manual processes in the 1980s, subsequent automation, pyrosequencing in the late 1990s and the development of next generation sequencing (NGS) in early 2000’s. However, all these sequencing technologies have been restricted to specialized laboratories, due to both the need for expensive equipment and the requirement of a high technical expertise. Furthermore, the features of these technologies, based on short readings, can limit the identification and characterization of structural variants (SVs) and haplotype analysis. The development of third-generation sequencing may solves some of these limitations and, a nanopore device (MinION), with small size, low cost and requirements takes a step forward in the concept of universalizing access to genomics.

Sequencing of a complete gene (SERPINC1: 7 exons / 13Kb) using NGS (PGM, Ion Torrent), Sanger (Abi3130xl, BigDye v3.0, AB) and nanopore (MinION, ONT) in 96 patients with antithrombin deficiency. For molecular analysis using the MinION we performed 3 overlapping long PCRs (LR-PCR, 8Kb, LongAmp, NEB) covering the whole SERPINC1 gene using DNA extracted from peripheral blood (QiAmp DNA Blood Mini kit, Qiagen). Barcodes (EXP-PBC001) were incorporated to each sample. Sequencing was run in a R9.4 flow cell with the ligation chemistry (SQK-LSK109). Bioinformatic analysis was performed using a specific pipeline developed by us that used available alignment, variant calling, structural variants and haplotyping public software. Additionally, in silico enrichment of chr1XXXXXXX region was also used to sequence unmanipulated genomic DNA of XX patients with SVs affecting SERPINC1 detected by other methods (MLPA, CGH, or LR-PCR).

SERPINC1 sequencing on the MinION device was the fastest (0.5h) and cheapest (0.0001€/bp) system. By being able to control the reading depth in real time, somatic variants can be detected. Furthermore, this system detects both SNVs and large indels and certain SVs. However, it showed limitations in the detection of small indels, especially those located in homopolymeric regions (a limitation that seems to be overcome with the development of new R10 flowcells). On the other hand, large SVs spanning the PCR primers are not detected. In contrast, long reads facilitated the identification of haplotypes for each allele. Finally, with in silico enrichment of unmanipulated genomic DNA in the region of the SERPINC1 gene we obtained 3-5X coverage after 48h of sequencing and a cost of 1000€/sample. Also that allowed us further SVs detection and epigenetic studies.

Our study shows a highly efficient sequencing system that allows universal sequencing processes due to its simplicity and cost. It requires an initial investment 100 times lower, has a costs 70 times lower, and render 8 times faster procedures than other sequencing systems. Finally, the possibilities of this equipment range from the identification of genetic variants, including SVs, to the determination of haplotypes and epigenetic studies, both for DNA and RNA.