Tequila-seq: Low-Cost Targeted Sequencing for Genomic Studies

Advancements in genomic sequencing have improved our ability to study genetic variations, but high costs remain a barrier. Tequila-seq is a cost-effective targeted sequencing method that enhances accessibility without sacrificing accuracy or efficiency.

By maintaining robust performance while lowering expenses, this approach allows researchers to investigate specific genomic regions affordably.

Fundamental Principles

Tequila-seq is based on targeted sequencing, which focuses on specific genomic regions rather than entire genomes. This method optimizes efficiency by directing sequencing resources to areas of interest, reducing costs while maintaining high coverage and accuracy. Unlike whole-genome sequencing (WGS), which generates vast amounts of data that may not always be relevant, targeted sequencing allows for deeper analysis of specific loci, improving variant detection and minimizing unnecessary sequencing.

A key feature of Tequila-seq is its use of multiplex PCR-based enrichment, which enables the simultaneous amplification of multiple target regions in a single reaction. This eliminates the need for expensive hybridization-based capture methods that require costly reagents and extensive processing. By leveraging a streamlined PCR approach, Tequila-seq achieves high specificity and uniformity across targeted regions, ensuring efficient sequencing read distribution. Studies have shown that PCR-based enrichment can achieve over 90% on-target read alignment, making it a reliable alternative to probe-based capture techniques.

Tequila-seq is also adaptable to different sequencing platforms, allowing researchers to integrate it into existing workflows without specialized equipment. It is compatible with both short-read and long-read sequencing technologies, providing flexibility based on study requirements. Short-read platforms like Illumina offer high accuracy and cost-effectiveness for detecting single nucleotide variants (SNVs) and small insertions or deletions (indels), while long-read platforms like Oxford Nanopore and PacBio enable the resolution of structural variants and complex genomic rearrangements. This versatility makes Tequila-seq suitable for applications ranging from clinical diagnostics to population genetics.

Key Components

Tequila-seq’s effectiveness relies on optimized reagents, targeted primer design, and sequencing platform compatibility. At its core is the multiplex PCR system, which requires carefully designed primers to amplify multiple genomic regions simultaneously. Success depends on minimizing primer-dimer formation and off-target amplification, which can reduce sequencing efficiency. Computational tools such as Primer3 and ThermoFisher’s Primer Designer help optimize primer selection for uniform amplification.

High-fidelity DNA polymerases, such as Q5 or KAPA HiFi, are preferred due to their low error rates and ability to accurately amplify GC-rich regions. These enzymes minimize polymerase-induced mutations, ensuring data integrity. Studies show that high-fidelity polymerases reduce PCR errors to below 1 × 10⁻⁶ per base pair, making them reliable for precise variant detection.

Uniform target enrichment ensures sequencing reads are evenly distributed across selected genomic regions. Without proper uniformity, some targets may be overrepresented while others receive insufficient coverage, leading to gaps in variant detection. Optimized primer concentrations and cycling conditions help balance amplification efficiency, improving sequencing depth consistency. Adjusting primer concentrations within a range of 0.1 to 0.5 µM enhances uniformity while preventing amplification bias.

Sequencing adapters also impact efficiency. Adapter ligation must be optimized to prevent chimera formation and adapter-dimer contamination, which can interfere with downstream data processing. Using uracil-containing adapters and enzymatic digestion of adapter dimers improves library quality, ensuring accurate read alignment. Additionally, unique molecular identifiers (UMIs) help distinguish true variants from PCR duplicates, increasing the reliability of variant calling in low-frequency mutation detection studies.

Steps In The Workflow

Tequila-seq follows a structured workflow to maximize efficiency and specificity in targeted sequencing. Each step, from library construction to sequencing, plays a crucial role in ensuring accurate variant detection and uniform coverage.

Library Construction

The process begins with preparing DNA libraries by fragmenting genomic DNA and adding sequencing adapters. Unlike whole-genome sequencing, which requires random fragmentation, Tequila-seq relies on targeted amplification, eliminating the need for mechanical shearing. High-quality DNA extraction is essential for consistent amplification, as degraded or contaminated samples can lead to uneven coverage.

Adapter ligation is optimized to minimize chimera formation, which can introduce sequencing artifacts. Enzymatic fragmentation-free approaches, such as direct PCR amplification from genomic DNA, streamline the process by reducing preparation time. Direct amplification methods improve library complexity while maintaining high on-target specificity. Incorporating unique molecular identifiers (UMIs) at this stage helps distinguish true variants from PCR duplicates, enhancing downstream variant calling accuracy.

Target Enrichment

After library construction, target enrichment is performed using multiplex PCR, allowing simultaneous amplification of multiple genomic regions in a single reaction. This step ensures that only regions of interest are sequenced, reducing unnecessary data generation and costs. Primer design is critical for enrichment efficiency, with computational tools aiding in optimizing specificity and minimizing off-target effects.

To achieve uniform amplification, primer concentrations and cycling conditions must be carefully adjusted. Balancing primer concentrations within a range of 0.1 to 0.5 µM significantly improves enrichment uniformity. High-fidelity polymerases, such as Q5 or KAPA HiFi, ensure accurate amplification with minimal errors, which is particularly important for applications requiring precise variant detection.

Amplification

Following target enrichment, additional PCR cycles generate sufficient material for sequencing. Over-amplification must be avoided, as it can introduce biases and reduce sequencing efficiency. Optimizing cycle numbers is crucial, as excessive amplification leads to PCR duplicates, reducing effective sequencing depth.

Reaction conditions such as annealing temperature and extension time are fine-tuned based on the target regions’ GC content. Adjusting annealing temperatures within a range of 55–65°C improves amplification efficiency while minimizing nonspecific products. Incorporating UMIs at this stage helps track duplicate reads, allowing for more accurate variant calling. Controlled amplification conditions ensure sequencing reads are evenly distributed across targeted loci, improving overall data quality.

Sequencing

The final step involves sequencing the enriched and amplified DNA libraries using a compatible platform. Tequila-seq works with both short-read and long-read sequencing technologies, providing flexibility based on study needs. Short-read platforms like Illumina offer high accuracy and cost-effectiveness for detecting SNVs and small indels, while long-read platforms like Oxford Nanopore and PacBio resolve structural variants and complex genomic rearrangements.

To maximize sequencing efficiency, library preparation must ensure optimal cluster generation or nanopore loading, depending on the platform. Adjusting input DNA concentrations within a range of 1–10 ng improves sequencing yield and data quality. Bioinformatics pipelines filter low-quality reads, remove PCR duplicates, and align sequences to reference genomes, ensuring accurate variant detection. By integrating these sequencing strategies, Tequila-seq provides a cost-effective yet reliable approach for targeted genomic studies.