Continuing with the Nanopore Learning course on Human genome sequencing and analysis, tonight I watched the video with the title “Library preparation: Ligation sequencing kit.” Hazel Johnson, a member of the Technical Services Team with Oxford Nanopore Technologies, explained the basics of library preparation for Nanopore sequencing. They defined library preparation as “converting sample into format compatible with the sequencing system” and this consists of attachment of “Y-shaped” sequencing adapters with the motor protein and leader sequence and the tether site. Johnson noted that the Ligation Sequencing Kit (LSK) is one of the most popular ones and takes about an hour. The input DNA amount is 1 ug or 100-200 fmol. The LSK is versatile and can produce 8+ Gbases on MinION/GridION flow cells and over 40 Gbases on PromethION flow cells. The kit has PCR and PCR-free barcoding options, which I didn’t know about! The LSK is versatile and can use any time of dsDNA: gDNA, cDNA, amplicons. The kit has reagents for six reactions. The expansion kits include native barcodes that are ligated directly onto templates with sufficient reagents for six libraries per barcode or the PCR barcoding expansion with barcodes introduced by PCR. Johnson noted that there is also a low input option EXP-PCA001 primers to amplify input material with adapters. With less than one microgram, ONT has used WGA RepliG to amplify libraries. The LSK XL kit can be automated or used with a multichannel pipette. The washkit can be used to remove >99.9% of library and reuse the flow cell. I will try the washkit for the 16S barcoding kit run we are doing in the BIT 477/577 Metagenomics course. This session helped me realize the barcoding options and versatility of the LSK.
