Belen de la Morena Barrio from the University of Murcia/CIBERER, Spain presented at London Calling 2022 on “Genetic dissection of structural variants in study subjects with antithrombin deficiency by nanopore sequencing.” Morena Barrio was introduced to Nanopore during their doctoral work, and they have now developed methods. They spoke about thrombin deficiency: antithrombin deficiency (SERPINC1) has 407 variants in the database! The disorder is explained by variants mostly in the encoding gene. However, other cases were structural variants that are >50 bp rearrangements, difficult to detect and characterize. Morena Barrio noted that nanopore sequencing was able to detect these variants. In this study, they recruited almost 400 subjects with antithrombin deficiency. Using MLPA, they identified duplications in SERPINC1 (n=4) and deletions in SERPINC1 (n=31).They also used Comparative Genome Hybridization (CGH) assays to detect other variants, identifying 26 deletions. CGH is based on probes and unable to detect specific break locations. They developed a long range PCR with Illumina NGS to identify breakpoints. In cases with longer variants, they performed Nanopore sequencing on the PromethION platform. They did this for 8 subjects. They also sequenced on a MinION on two samples. Finally, they used adaptive sampling for some samples. Morena Barrio explained that they developed a bioinformatic analysis pipeline with alignment (minimap2) and variant calling with sniffles. Discrepancies between MLPA, LR+PCR/NGS were resolved with Nanopore sequencing. Nanopore sequencing of two subjects identified the insertion of 2.4 kb. With just 15 minutes of sequencing, they obtained enough sequences pero sample for analysis.Morena Barrio summarized that nanopore sequencing has “resolution” and concluded that Nanopore sequencing allows identification of variants.
