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Updated: May 12, 2026
10:24
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
Quantifying sequence proportions in a DNA-based diet study using Ion Torrent amplicon sequencing: which counts count?
Bruce E Deagle1, Austen C Thomas, Amanda K Shaffer
1Australian Antarctic Division, Channel Highway, Kingston, Tas, Australia. bruce.deagle@aad.gov.au
Molecular Ecology Resources
|April 18, 2013
Summary
Environmental DNA (eDNA) sequence counts from seal scat show significant biases. Careful bioinformatic analysis and experimental design are crucial for accurate dietary quantification in eDNA studies.
Area of Science:
- Ecology
- Genetics
- Bioinformatics
Background:
- Environmental DNA (eDNA) barcoding aims to quantify taxa abundance from sequence reads.
- Potential biases in high-throughput sequencing for quantitative eDNA analysis are not fully understood.
Purpose of the Study:
- To investigate biases in using sequence read proportions from DNA barcoding to quantify the diet of harbour seals (Phoca vitulina).
Main Methods:
- DNA was amplified from captive harbour seal scats using a 16S mitochondrial marker.
- Amplicons were sequenced using Ion Torrent PGM™, with varying bioinformatic parameters.
- Prey proportions in scats were compared to known dietary mass proportions.
Main Results:
- Sequence proportions showed low inter-sample variability for a given parameter set.
- Proportions were sensitive to sequencing direction, quality filtering, read length, and primer tags.
- Less stringent filtering yielded more consistent data but still differed from dietary mass, indicating biases.
Conclusions:
- Quantitative interpretation of eDNA sequence proportions requires rigorous experimental design and data analysis.
- Technical and biological biases can significantly affect the accuracy of eDNA-based dietary quantification.
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