Nanopore sequencing has been widely used for the real-time detection and surveillance of pathogens with portable MinION. Nanopore adaptive sequencing can enrich on-target sequences without additional pretreatment. In this study, the performance of adaptive sequencing was evaluated for viral genome enrichment of clinical respiratory samples. Ligation-based nanopore adaptive sequencing (LNAS) and rapid PCR-based nanopore adaptive sequencing (RPNAS) workflows were performed to assess the effects of enrichment on nasopharyngeal swab samples from human adenovirus (HAdV) outbreaks. RPNAS was further applied for the enrichment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swab samples to evaluate sensitivity and timeliness. The RPNAS increased both the relative abundance (7.87–12.86-fold) and data yield (1.27–2.15-fold) of HAdV samples, whereas the LNAS increased only the relative abundance but had no obvious enrichment on the data yield. Compared with standard nanopore sequencing, RPNAS detected the SARS-CoV-2 reads from two low-abundance samples, increased the coverage of SARS-CoV-2 by 36.68–98.92%, and reduced the time to achieve the same coverage. Our study highlights the utility of RPNAS for virus enrichment directly from clinical samples, with more on-target data and a shorter sequencing time to recover viral genomes. These findings promise to improve the sensitivity and timeliness of rapid identification and genomic surveillance of infectious diseases.

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IntroductionNanopore sequencing has been widely used in clinical metagenomic sequencing for pathogen detection with high portability and real-time sequencing. Oxford Nanopore Technologies has recently launched an adaptive sequencing function, which can enrich on-target reads through real-time alignment and eject uninteresting reads by reversing the voltage across the nanopore. Here we evaluated the utility of adaptive sequencing in clinical pathogen detection.MethodsNanopore adaptive sequencing and standard sequencing was performed on a same flow cell with a bronchoalveolar lavage fluid sample from a patient with Chlamydia psittacosis infection, and was compared with the previous mNGS results.ResultsNanopore adaptive sequencing identified 648 on-target stop receiving reads with the longest median read length(688bp), which account for 72.4% of all Chlamydia psittaci reads and 0.03% of total reads in enriched group. The read proportion matched to C. psittaci in the stop receiving group was 99.85%, which was much higher than that of the unblock (<0.01%) and fail to adapt (0.02%) groups. Nanopore adaptive sequencing generated similar data yield of C. psittaci compared with standard nanopore sequencing. The proportion of C. psittaci reads in adaptive sequencing is close to that of standard nanopore sequencing and mNGS, but generated lower genome coverage than mNGS.DiscussionNanopore adaptive sequencing can effectively identify target C. psittaci reads in real-time, but how to increase the targeted data of pathogens still needs to be further evaluated.