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

Daniela Gaio1, Kay Anantanawat1, Joyce To1

  • 1iThree Institute, University of Technology Sydney, Sydney, NSW, Australia.

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|January 11, 2022
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Summary
This summary is machine-generated.

Researchers can now generate up to 14 times more sequencing libraries for the same cost using the new Hackflex method. This low-cost approach ensures high-quality library preparation for Illumina sequencing, making large-scale genomics projects more accessible.

Keywords:
Illumina sequencingNextera Flexhigh-throughput sequencinglibrary preparationmetagenomicsmultiplex

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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.