When to use next generation sequencing or diagnostic PCR in diet analyses

Area of Science:

• Ecology
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) is widely used for DNA-based diet analysis.
• Diet samples often contain mixed DNA from prey and consumers, complicating NGS analysis.
• Consumer DNA can reduce the accuracy of food DNA detection in NGS.

Purpose of the Study:

• To compare the reliability of NGS and diagnostic PCR for diet analysis in predatory beetle regurgitates.
• To determine the required sequencing depth or replication for stable results with NGS and diagnostic PCR.
• To assess if general barcoding primers co-amplifying consumer DNA affect prey detection.

Main Methods:

• Investigated predatory beetle regurgitates with varying DNA mixtures.
• Compared prey DNA detection using NGS and diagnostic PCR.
• Assessed the impact of consumer DNA co-amplification on NGS results.
• Evaluated sequencing depth and replication needs for both methods.

Main Results:

• NGS showed variability in prey detection across repeated sample analyses, indicating incomplete diet description.
• Diagnostic PCR detected prey sequences missed by NGS, especially when consumer DNA was present.
• High sequencing depth is required for reliable NGS diet analysis in complex samples, impacting cost-efficiency.

Conclusions:

• Diagnostic PCR provides more reliable and consistent prey detection than NGS for complex diet samples.
• PCR is more cost-effective and suitable for large-scale diet studies, particularly for ecologically important prey.
• NGS is better for discovering the range of prey but less reliable for comparing species co-occurrences.