Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• High-throughput sequencing requires efficient and cost-effective library preparation.
• Existing methods like Nextera Flex (Illumina DNA Prep) can be expensive for large-scale projects.
• The need for affordable, high-quality sequencing library production is critical for advancing genomic research.

Purpose of the Study:

• To develop and validate a novel, low-cost method for producing Illumina-compatible sequencing libraries.
• To assess the quality and performance of the new method, termed Hackflex, compared to standard protocols.
• To demonstrate the cost-effectiveness and scalability of Hackflex for high-throughput sequencing applications.

Main Methods:

• Developed Hackflex, a modified library preparation protocol for Illumina sequencing.
• Tested Hackflex using genomic DNA from *Escherichia coli*, *Pseudomonas aeruginosa*, and *Staphylococcus aureus*.
• Compared Hackflex performance against standard Nextera Flex (Illumina DNA Prep) and a diluted transposase variation.
• Evaluated library quality, coverage uniformity, and yield, including size-selected libraries for *de novo* assembly.

Main Results:

• Hackflex produced high-quality libraries with uniform coverage comparable to standard Nextera Flex.
• Size-selected Hackflex libraries yielded sufficient complexity for *de novo* microbial genome assembly.
• Assemblies from large-insert Hackflex libraries were more contiguous than those from standard libraries.
• Hackflex achieved a per-sample reagent cost of A$7.22, representing a 9.87-fold reduction compared to standard Nextera Flex.
• A simplified Hackflex protocol achieved a 14-fold overall cost saving.

Conclusions:

• Hackflex is a cost-effective and high-quality method for Illumina sequencing library preparation.
• The method enables researchers to generate significantly more libraries within budget, increasing data output.
• Hackflex facilitates large-scale genomic studies and improves *de novo* genome assembly contiguity, especially with size selection.