A practical method for barcoding and size-trimming PCR templates for amplicon sequencing.

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Next-generation sequencing (NGS) enables high-throughput analysis of numerous samples.
• Current methods for barcoding and size-trimming long PCR products in NGS workflows require optimization for efficiency.
• Accurate targeting of DNA regions is crucial for robust phylogenetic studies.

Purpose of the Study:

• To develop a more efficient method for high-throughput DNA barcoding and size-trimming of long PCR products for NGS.
• To present a novel two-step PCR approach for DNA template preparation.
• To enhance phylogenetic analyses through improved DNA region targeting.

Main Methods:

• A two-step PCR strategy was employed for sample barcoding.
• Following barcoding, a pool shearing, adapter ligation, and 5' end selection protocol was utilized.
• This method allows for the size-trimming of DNA templates of varying lengths.

Main Results:

• The developed method enables efficient barcoding and size-trimming for large sets of DNA samples.
• The protocol is effective for DNA templates of any size.
• The approach facilitates precise targeting of specific DNA regions.

Conclusions:

• The new trimming method provides significant advantages for phylogenetic studies.
• Consistent targeting of DNA regions maximizes sequence alignment quality.
• This technique supports the effective application of operational taxonomic unit (OTU)-based algorithms in phylogenetic research.