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:Library preparation protocol for 16s or ITS
Sequencing
We are sequencing using Illumina instruments, which all use a common chemistry. They differ some in the nature of the flow cells. The following video gives an overview of the sequencing by synthesis that happens on the Illumina instrument and would apply to the sequencing of one or more of our sequencing libraries. We have an iSeq device at UW for small scale work, and use sequencers elsewhere (e.g., a NovaSeq instrument at the GSAF at the University of Texas) for production work.
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