An efficient and cost-effective method for primer-induced nucleotide labeling for massive sequencing on...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) is crucial for large DNA database creation, such as DNA barcoding.
• Simultaneous sequencing of numerous samples on NGS platforms presents a challenge.

Purpose of the Study:

• To develop and demonstrate a primer-induced sample labeling method for high-throughput sequencing of many samples.
• To enable efficient construction of large DNA reference libraries.

Main Methods:

• A strategy was developed to label samples with unique oligonucleotides (oligos) attached to primer 5'-ends.
• 894 samples were labeled using unique pentanucleotide oligos on primers for ITS, matK, and rbcL amplification.
• Pooled PCR products from labeled samples were sequenced on a high-throughput platform.

Main Results:

• Successful sequencing rates of 87.02% (rbcL), 89.15% (matK), and 95.53% (ITS) were achieved.
• The mean label mismatch ratio was 5.68%.
• A sequencing depth of 30×–40× was sufficient for reliable sequence data.

Conclusions:

• The primer-induced sample labeling method is easy, reliable, and cost-efficient for large-scale sample sequencing.
• This technique is adaptable for labeling any number of samples by adjusting oligo length.
• The method is valuable for DNA barcoding, population biology, and community phylogenetics reference library construction.