A broadly applicable COI primer pair and an efficient single-tube amplicon library preparation protocol for...

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• The cytochrome c oxidase subunit I (COI) gene is crucial for species identification but its variability complicates universal primer design for metabarcoding.
• Existing primers like mCOIintF are optimized for aquatic environments, limiting terrestrial species coverage.
• Metabarcoding library preparation often involves multiple steps, increasing complexity and potential for bias.

Purpose of the Study:

• To develop novel, highly-covering primers for COI gene amplification in terrestrial metabarcoding.
• To create a streamlined, single-tube protocol for library preparation, integrating amplification, MIDs, and adapter ligation.
• To evaluate the efficiency and bias of the new primers and protocol in mock community sequencing.

Main Methods:

• Design of a new forward primer, Sauron-S878, targeting improved terrestrial COI sequence coverage.
• In silico analysis of primer coverage against NCBI sequences, comparing Sauron-S878 with mCOIintF.
• In vitro amplification testing with 255 DNA extracts and subsequent sequencing using a single-tube protocol with Illumina MiSeq.

Main Results:

• Sauron-S878 increased in silico COI sequence coverage from 74.4% to 98.3% compared to mCOIintF.
• The primer pair Sauron-S878/jgHCO2198 amplified 98.4% of tested DNA extracts, outperforming other common COI primers.
• The single-tube protocol successfully generated libraries, recovering 75% of taxa from mock samples without significant bias, though DNA concentration influenced read counts.

Conclusions:

• The Sauron-S878/jgHCO2198 primer pair and single-tube protocol offer enhanced coverage and simplified library preparation for terrestrial metabarcoding.
• The developed method demonstrates high efficiency and flexibility, comparable to commercial kits.
• Potential biases related to DNA template concentration in metabarcoding require further investigation for accurate biodiversity assessment.