Objective To evaluate the basic performance and clinical application value of nanopore sequencing, in order to provide new ideas for the rapid detection of clinical etiology. Methods From December 2021 to May 2022, blood samples from inpatients suspected of bloodstream infection in Renmin Hospital of Wuhan University were collected, and the nanopore sequencing platform and blood culture method were used to simultaneously identify the pathogenic bacteria in the blood samples of the selected patients, and identify the pathogenic bacteria in the blood samples of the selected patients. The basic performance and clinical utility of nanopore sequencing were evaluated. Results A total of 251 patients were included, and 119 patients (47.4%) were found to have pathogens by nanopore sequencing, which was higher than that of 23 patients (9.2%) by blood culture (χ2=79.167, P<0.001). The results of the two methods are not consistent (kappa=0.052, P=0.175). Nanopore sequencing has a certain missed detection rate. In terms of the types of pathogenic bacteria detected, 47 bacteria and 15 fungi were detected by nanopore sequencing. Conclusion Compared with blood culture, nanopore sequencing has a higher detection rate and more types of pathogens. This technology has obvious advantages in the rapid diagnosis of bloodstream infection pathogens.
Objective To explore a new method of extending circulating tumor DNA (ctDNA) by adapter ligation to adapt it to nanopore sequencing. Methods A RC adapter was designed to extend ctDNA fragments, and reaction conditions including end repair, dA-tailing, and ligation were optimized. A dual-barcode and RC-adapter-based sample splitting workflow was established, leading to the development of a novel nanopore sequencing based method for ctDNA methylation detection, named RCnano. Results Agarose gel electrophoresis and sequencing results showed that the 48 bp adapter was the optimal length for RCnano. Secondary sample splitting based on RC adapter sequences recovered 42% of unclassified reads, equivalent to a 4%-5% increase in total data yield. Compared with standard nanopore sequencing library preparation, the optimized RCnano workflow increased sequencing output by approximately 6-fold. Among four nanopore methylation callers, Nanopolish performed best, with a mean absolute error of 0.021. RCnano showed good correlation with bisulfite amplicon sequencing (r2=0.952) and was able to detect methylation sites at an abundance as low as 0.1%. Conclusions This study demonstrates a novel application of nanopore sequencing for ultrasensitive ctDNA methylation analysis, which could enhance noninvasive cancer diagnostics and real-time epigenetic monitoring.
