A method to generate multilocus barcodes of pinned insect specimens using MiSeq

Area of Science:

• Molecular Biology
• Genomics
• Entomology

Background:

• Amplicon sequencing is cost-effective for molecular insect identification, but short reads from platforms like Illumina MiSeq can lead to incomplete or chimeric sequences.
• Overlapping short gene fragments requires complex bioinformatics, increasing the risk of errors in molecular identification.
• Existing methods struggle with degraded DNA from older museum specimens.

Purpose of the Study:

• To develop a modified nested amplicon sequencing method for generating high-quality, near full-length DNA barcodes from pinned insect specimens.
• To overcome limitations of short-read sequencing for insect identification using museum collections.
• To improve the reliability of multilocus barcoding for entomological research.

Main Methods:

• A three-step nested PCR approach was developed to target near full-length loci.
• Short fragments (300-350 bp) were amplified for high-throughput sequencing on the Illumina MiSeq platform.
• The method was tested on pinned insect specimens, including bees, with varying preservation ages.

Main Results:

• The modified method successfully generated 407 sequences of three loci from 86% of specimens.
• 71% of pinned bee specimens met the 95% sequence similarity threshold for species replicates.
• The method showed optimal performance for specimens aged 0-5 years, with limitations for older samples (10 years pinned, 14 years ethanol-preserved).

Conclusions:

• The modified nested amplicon sequencing method effectively addresses challenges associated with short-read next-generation sequencing.
• This technique enhances the ability to create high-quality multilocus barcodes from insect collections, particularly valuable for molecular identification.
• The method provides a robust tool for utilizing historical insect specimens in molecular research.