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Updated: Mar 7, 2026
10:24
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
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High-throughput sequencing of multiple amplicons for barcoding and integrative taxonomy
Perrine Cruaud1, Jean-Yves Rasplus1, Lillian Jennifer Rodriguez1,2
1INRA, UMR1062 CBGP, F-34988 Montferrier-sur-Lez, France.
Scientific Reports
|February 7, 2017
Summary
Next-generation sequencing (NGS) offers potential for DNA barcoding and integrative taxonomy. This study developed a workflow for quality control, showing NGS can efficiently sequence multiple markers for species identification.
Area of Science:
- Molecular Biology
- Taxonomy
- Bioinformatics
Background:
- Next-generation sequencing (NGS) potential for DNA barcoding and integrative taxonomy remains largely unrealized.
- Previous DNA barcoding efforts primarily relied on Sanger sequencing.
Purpose of the Study:
- To assess the feasibility of using Illumina MiSeq for constructing DNA barcode libraries.
- To develop and validate a quality control workflow for NGS data in taxonomy.
- To compare NGS data with existing Sanger sequences for species identification.
Main Methods:
- Amplification of multiple markers from hundreds of fig wasp specimens using a two-step PCR.
- Sequencing using Illumina MiSeq platform.
- Development of a data quality control workflow.
- Comparison of Illumina and Sanger sequences.
Main Results:
- Primers and PCR conditions for Sanger sequencing were directly applicable to MiSeq library construction.
- After quality control, 87% of species and 76% of specimens yielded valid MiSeq sequences.
- Major sequence clusters did not always align with targeted genetic loci.
- Nine specimens showed two divergent sequences (up to 10%).
- 95% of species had similar MiSeq and Sanger sequences; remaining 5% indicated potential NUMTs or heteroplasmy.
Conclusions:
- Illumina sequencing, while powerful, is not free from artifacts, necessitating rigorous quality control.
- Existing Sanger databases may contain non-target genes, emphasizing the need for careful data validation.
- Utilizing multiple markers and collaborating with taxonomists are crucial for accurate DNA taxonomy and species diversity assessment.
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