Addressing nontarget amplification in DNA metabarcoding studies of arthropod-feeding rodents

Area of Science:

• Ecology
• Genetics
• Molecular Biology

Background:

• High-throughput sequencing and DNA metabarcoding are revolutionizing animal diet studies.
• Mitochondrial cytochrome oxidase C subunit I (mtCOI) metabarcoding is common for arthropod diet analysis.
• Nontarget amplification of consumer DNA is a significant challenge in metabarcoding.

Purpose of the Study:

• To investigate the impact of nontarget amplification on arthropod diet analysis in woodrats (genus *Neotoma*).
• To provide guidance on identifying and reducing nontarget amplification in DNA metabarcoding studies.
• To develop a resource for assessing nontarget amplification risk in rodent diet studies.

Main Methods:

• Examined diets of *Neotoma* woodrats in natural and captive environments.
• Analyzed DNA metabarcoding data to quantify nontarget amplification effects.
• Developed a database of mtCOI sequence mismatches for rodents and common primers.

Main Results:

• Nontarget amplification significantly reduced arthropod DNA reads in woodrat fecal samples.
• This complication inhibited downstream analyses of dietary diversity and composition.
• A database was created to assess the risk of nontarget amplification in rodent systems.

Conclusions:

• Nontarget amplification is a critical issue in arthropod DNA metabarcoding, particularly in rodents.
• The study offers practical strategies to minimize nontarget amplification for more accurate dietary assessments.
• The developed resource aids researchers in designing robust metabarcoding studies for rodent diets.