Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform

Area of Science:

• Genomics
• Biosystematics
• Molecular Biology

Background:

• DNA barcoding is crucial for specimen identification in biosystematics.
• Sanger sequencing remains common for DNA barcoding despite advances in High-Throughput Sequencing (HTS).

Purpose of the Study:

• To develop a scalable double dual-indexing approach for sequencing DNA barcode markers using Illumina Miseq.
• To evaluate the efficiency, cost-effectiveness, and data recovery of this HTS method for arthropod identification.

Main Methods:

• Utilized a double dual-indexing strategy on an Illumina Miseq platform.
• Sequenced the cytochrome c oxidase I (COI) DNA barcode marker for 1,010 arthropod specimens.
• Analyzed success rate, sequence recovery, cost, and labor time.

Main Results:

• Achieved a 97.3% success rate in sequencing 658 base pairs of the COI barcode.
• Recovered a higher proportion of DNA barcode sequences per specimen compared to Sanger sequencing.
• Reduced per-specimen costs and labor time by nearly 80%.

Conclusions:

• The presented HTS double dual-indexing method is a scalable and cost-effective solution for DNA barcoding.
• This approach enhances genetic variation analysis by enabling recovery of multiple sequences per specimen.
• Offers a significant improvement over traditional Sanger sequencing for large-scale specimen identification.