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Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20th compared to conventional Illumina methods. Copyright: © 2023 Jones et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Ashley Jones, David Stanley, Scott Ferguson, Benjamin Schwessinger, Justin Borevitz, Norman Warthmann. Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing. PloS one. 2023;18(1):e0280004

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PMID: 36706059

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