RESEARCH ARTICLE Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes

Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequenc-ing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly re-agents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nex-tera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Sev-eral hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chro-mosomes, and RNA sequencing