Preparation of Illumina 16s Amplicon Sequencing Libraries with Peptide Nucleic Acids (PNAs) for the Analysis of Maize-Associated Microbiomes
- 1Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia 30602, USA
- 2Department of Crop and Soil Science, University of Georgia, Athens, Georgia 30602, USA
- 3Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, Georgia 30602, USA
- ↵4Correspondence: jason.wallace{at}uga.edu
Abstract
One of the most common methods to survey bacterial communities is targeted amplification of the hypervariable regions of the 16s rRNA gene followed by sequencing. This protocol details Illumina library preparation of such amplicons from communities isolated from maize. We include both staggered PCR primers to improve Illumina base calling and peptide nucleic acids (PNAs) to reduce the presence of plant organelles. Primers are designed with Illumina adapter sequences for the addition of sample-specific indexes (barcodes). We also briefly discuss alternative primer sets, including ones that directly discriminate against plant organelles or that amplify different organisms (e.g., fungal internal transcribed spacer [ITS] sequences).
Footnotes
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From the Maize collection, edited by Candice N. Hirsch and Marna D. Yandeau-Nelson. The entire Maize collection is available online at Cold Spring Harbor Protocols and can be accessed at https://cshprotocols.cshlp.org/.
