CUPID-seq enables highly multiplexed amplicon sequencing via combinatorial in-line dual indexing.

Area of Science:

• Genomics
• Microbial Ecology
• Molecular Biology

Background:

• Targeted amplicon sequencing is crucial for genetic variation profiling, particularly in microbial ecology using 16S/18S rRNA genes.
• High-capacity sequencing platforms are limited by unique dual indexes (UDIs), increasing costs and reducing sample pooling capacity.

Purpose of the Study:

• To introduce CUPID-seq, a scalable amplicon sequencing strategy.
• To reduce costs and increase throughput for targeted sequencing applications.

Main Methods:

• CUPID-seq employs combinatorial indexing across two PCR rounds with phased, in-line UDIs introduced during Round 1.
• This allows multiple samples to share Illumina UDIs in Round 2 while maintaining unique identification.
• Primers targeting the 16S V4 region were developed and validated, alongside a computational demultiplexing workflow.

Main Results:

• CUPID-seq reduces upfront costs by up to 85% and library preparation time and reagent use by up to 40%.
• The method enables unique sample identification even when sharing Illumina UDIs.
• Validated for 16S rRNA gene sequencing, adaptable to other amplicons.

Conclusions:

• CUPID-seq enhances the scalability of amplicon sequencing.
• It lowers costs and increases multiplexing capacity, improving the efficiency of high-throughput sequencing platforms.
• The strategy is broadly applicable across diverse biological research areas.