DNA Metabarcoding Using Indexed Primers: Workflow to Characterize Bacteria, Fungi, Plants, and Arthropods from Environmental Samples

• NCJ Number: 310322
• Journal: Diversity Volume: 17 Issue: 2 Pages: 137
• Date Published: February 2025
• Author(s): Teresa M. Tiedge; Kelly A. Meiklejohn
• Length: 18 pages
• Grant Number(s): 2020-R2-CX-0035
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)

Annotation

This article discusses DNA metabarcoding using indexed primers.

Abstract

The study sought to develop a wet laboratory workflow utilizing indexed primers that could cost-effectively reduce bench time while simultaneously targeting multiple DNA barcode regions to characterize bacterial (16S), fungal (ITS1), plant (ITS2, trnL p6 loop), and arthropod (COI) communities. The Earth Microbiome Project (EMP) adopted the use of indexed primers, PCR primers containing Illumina^® adapter sequences and a unique 12-nucleotide Golay barcode to simplify the identification of bacterial taxa via the 16S barcode. For environmental samples, sequencing was successful for all primers except COI c, and primer biases were observed for all three plant primers, in which a small number of families were uniquely amplified for each primer pair. This workflow can be applied to many disciplines that utilize DNA metabarcoding given its customizability and flexibility with Illumina sequencing chemistry. The EMP primer constructs for 16S were modified to accommodate the DNA barcode regions of interest while also permitting successful demultiplexing following sequencing. A single indexed primer pair was designed for ITS1 and trnL p6 loop, and two primer pairs were developed for ITS2 and COI. To test the workflow, a total of 648 soil and 336 dust samples were processed, with key steps including DNA isolation, total DNA quantification, amplification with indexed primers, library purification and quantification, and Illumina MiSeq sequencing. The combination of the three plant targets successfully recovered all plant taxa in the positive controls except for Nephrolepis exaltata [Nephrolepidaceae] and the COI primers recovered all arthropod taxa except for the beetle. Notably, none of the taxa in the fungal positive control were recovered using ITS1. (Published Abstract Provided)