# Rapid Nanopore Sequencing of Positive Blood Cultures Using Automated Benzyl-Alcohol Extraction Improves Time-Critical Sepsis Management

## TL;DR

A new automated method using benzyl alcohol improves nanopore sequencing of blood cultures, speeding up sepsis diagnosis.

## Contribution

Integration of benzyl alcohol SPS removal into an automated extractor enhances nanopore sequencing for rapid pathogen detection.

## Key findings

- Automated extraction produced purer DNA with higher A260/A280 and A260/A230 ratios compared to manual methods.
- The automated workflow generated more reads and longer read lengths, improving species classification accuracy.
- The sample-to-answer time was reduced to under 8 hours, making it suitable for critical care settings.

## Abstract

Background/Objective: Timely identification of bloodstream pathogens is critical for sepsis management; however, PCR inhibitors such as sodium polyanetholesulfonate (SPS) in blood culture broth compromise nucleic acid recovery and long read sequencing. We assessed whether coupling a benzyl alcohol SPS-removal step to the fully automated LabTurbo AIO extractor improves Oxford Nanopore-based pathogen detection. Methods: Thirteen positive blood culture broths were pre-treated with benzyl alcohol and divided: half volumes were purified on the LabTurbo AIO; paired aliquots underwent manual QIAamp extraction. DNA purity was evaluated by NanoDrop and Qubit. Barcoded libraries were sequenced on MinION R9.4.1 flow cells for 6 h. Results: Automated eluates showed a median A260/A280 of 1.92 and A260/A230 of 1.96, versus 1.80 and 1.48 for manual extracts. The automated workflow generated 1.69 × 106 total reads compared with 3.9 × 105 reads for manual extraction. The median N50 read length increased from 5.9 kb to 8.7 kb, and the median proportion of reads classified to species increased from 62% to 84%. The hands-on time was <5 min and the sample-to-answer turnaround was <8 h, compared with >9 h and 90 min for the manual protocol, respectively. Conclusions: Benzyl alcohol SPS removal integrated into the LabTurbo AIO extractor yielded purer, longer, and higher read counts, enhancing nanopore sequencing depth and accuracy while compressing diagnostic turnaround to a single working day. This represents a practical advance for rapid blood culture pathogen identification in critical care settings.