Tonight I continued watching the Nanopore Learning Metagenomics series. The lessons focused on Library Preparation: Low input PCR kits. Hazel Johnson spoke about low-input kits. Library preparation involves attachment of sequencing adapters that are “Y” shaped and include a motor protein and leader sequence. A tether site facilitates the capture of the molecule. When you have less than 100 ng of input, there are two PCR options: PCR sequencing kit and Rapid PCR barcoding kit. PCR selects for molecules with the adapter. The Rapid PCR kit contains twelve barcodes. During PCR, the primers have 5′ tags that enable attachment of the sequencing adapters. The Rapid PCR Barcoding kit produces 2-4 Kb products. Specific output can be sequenced. Whole genome amplification is recommended for input less than 1 ng. The WGA kits from QIAGEN offer options for traditional and ultra fast amplification with scalable output. Johnson explained that the 16 barcoding kit can be used for bacterial genus amplification. The 16S rRNA gene can be analyzed for phylogenetic reconstruction. Johnson noted that this kit is useful in cases with potential host DNA “contamination.” Ten nanograms of input is the minimum. There is one bead cleanup step to exchange the buffer and prepare for sequencing. The EPI2ME workflow can be used for analysis. A report is generated in real-time with reads classified and phylogenetic tree output. We will use this kit in lab next week. I will check what updates have been added to EPI2ME 16S workflows.
