Amplicon libraries
We use PCR to amplify 16S and ITS regions and attach unique indexes (or barcodes) to each product. We then pool the products into a library. In the future we will also develop the ability to target other loci (e.g., CO1 in animals).
Linda van Diepen's powerpoint presentation with information about the steps in isolating DNA, PCR amplification, and bioinformatics.
We are targeting the V4 region of the 16S ribosomal subunit, which is encoded by DNA. The transcribed 16S RNA molecule looks like this (when self-complementary regions in the RNA sequence fold onto themselves to make stems), which highlights conserved, functional regions (the stems) and more variable intervening regions in the loops (or otherwise labeled as variable). The image is in the public domain and is from the 16S wikipedia page (which has additional cool and relevant information, including primer sequences and information about the V4 region we target).
Our Protocol:16S and/or ITS Library Prep Protocol