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Among nucleic acid investigating methods, high-throughput sequencing has become one of the most important tools for both research and diagnostics. Determining the genome sequence of a microorganisms confirms the diagnostic results, and helps to understand the genome structure and follow genetic changes of the pathogen.

Numerous kits are available for the extraction of nucleic acid and preparation of DNA libraries. Systems suitable for generating long sequences (long read sequencing) are more sensitive to nucleic acid fragmentation than those designed for producing short sequences (short read sequencing), so great care must be taken when selecting the isolation technology.

The aim of our study was to select the most suitable DNA extraction kits for preparing long read sequencing of bacterial genomes. For this purpose, we tested two manual kits (the filter column-based Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit and the liquid centrifugation-based InviPrep Fast Lysis Buffer) and two automated, magnetic bead-based isolation system kits (MagCore Genomic DNA Bacterial Kit and MagnifiQ 1 Genomic DNA instant kit). Based on literature data and our laboratory experience, the Zymo kit is suitable for long read sequencing preparation, so it served as the basis for comparison. Pseudomonas aeruginosa bacterial strains (n=6) propagated in liquid culture were dispensed in their ideal growth phase, and following centrifugation the pellets were used for extraction. The concentration of the eluates was measured, then DNA library was prepared, and sequencing was performed using the Nanopore Native Barcoding kit and MinION mk1b device. After bioinformatics analysis, mean read length, mean read quality, the read count, total bases and read length N50 were compared.

Although extraction with the Zymo kit took increased hands-on time, it proved to be the most ideal in terms of read length. The highest DNA concentration was produced with the MagCore kit, but the read length was lower than with the former. In the case of the other two kits, both the nucleic acid concentration and read length were lower than those achieved with the Zymo kit.

Although all three DNA purification methods involve shorter protocols than the Zymo Kit, they are not ideal for DNA extraction prior to long read sequencing. Since the DNA concentration and fragment size obtained with the previously used kit are suitable for library preparation, we will use this for our research projects planned. The suitability of newly developed isolation methods will be continuously evaluated.