Rapid detection of single nucleotide polymorphisms using the MinION nanopore sequencer: a feasibility study for perioperative precision medicine

Introduction: Precision medicine is a phrase used to describe personalized medical care tailored to specific patients based on their clinical presentation and genetic makeup. However, despite the fact that several single nucleotide polymorphisms (SNPs) have been reported to be associated with increased susceptibility to particular anesthetic agents and the occurrence of perioperative complications, genomic profiling and thus precision medicine has not been widely applied in perioperative management. Methods: We validated six SNP loci known to affect perioperative outcomes in Japanese patients using genomic DNA from saliva specimens and nanopore sequencing of each SNP loci to facilitate allele frequency calculations and then compared the nanopore results to those produced using the conventional dideoxy sequencing method. Results: Nanopore sequencing reads clustered into the expected genotypes in both homozygous and heterozygous cases. In addition, the nanopore sequencing results were consistent with those obtained using conventional dideoxy sequencing and the workflow provided reliable allele frequency estimation, with a total analysis time of less than 4 h. Conclusion: Thus, our results suggest that nanopore sequencing may be a promising and versatile tool for SNP genotyping, allowing for rapid and feasible risk prediction of perioperative outcomes.

Identification and Confirmation of Chironomus circumdatus Kieffer, 1916 (Diptera: Chironomidae) Using DNA Barcoding

Present study was performed to identify and confirm collected larval Chironomid up to species level. To assess molecular taxonomy and phylogeny, DNA barcoding was done using Sanger dideoxy sequencing of mitochondrial cytochrome c oxidase subunit I (COI) gene of larval sample. Bioinformatics analysis was done by using NCBI’s BLAST software. By evidence of DNA barcoding, it was confirmed that present larval chironomid species was Chironomus circumdatus Kieffer, 1916.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda

Background In Uganda artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine (SP) for chemoprevention during pregnancy, but drug resistance may limit efficacies. Methods Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and qPCR assays. Results Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of PfCRT 76T decreased, but varied markedly between sites (0-48% in 2018; 0-22% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For PfMDR1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14/16 sites, and gene amplification was not seen. Considering mutations associated with high level SP resistance, prevalences of PfDHFR 164L (up to 80%) and PfDHPS 581G (up to 67%) were high at multiple sites. Considering PfK13 propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 40%, respectively). Conclusions We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

Screening forBRCAmutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of theBRCAgenes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.