Tonight I continued watching the Nanopore Learning sessions about Metagenomics. The Metagenomics lessons I started with were “Library Preparation: An introduction and overview of the options available.” Hazel Johnson provided an overview of the library preparation protocols and kits offered. They defined library preparation as “converting sample into format compatible with the sequencing system” by attaching the Y-shaped sequencing adapter with the motor protein and leader sequence and a tether site. All library preparation kits have the same final configuration with sequencing adapter on the far ends of the DNA molecule. Library preparation options include manual, automated and ubiquitous. The rapid kits prepare libraries using a two step transposase system. These kits generate random population of fragments. The ligation sequencing kits are engineered for maximum throughput. For RNA, there are direct RNA and cDNA sequencing options. RNA sequencing provides information of modified bases. If input is low, the PCR cDNA sequencing kit is available. Both kits can be used for multiplexing samples. The VolTRAX2 has cartridges for automated library prep. Library preparation using PCR-free kits adds the adapter and tether site to the template DNA molecules. The ligation sequencing kit prepares DNA by repairing ends and ligating sequencing adapters. This kit does require a bead cleanup and is engineered for read length and throughput. The rapid sequencing kit takes only ten minutes and uses transposome complex for adapter addition. To decide which kit to use, Johnson suggested considering speed, throughput, read length, and preservation of modifications. The details provided by these videos helped learn about adapter ligation and throughput considerations.
