title: Inexpensive multiplexed library preparation for megabase-sized genomes.
abstract: 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 sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents 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 Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several 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 chromosomes, and RNA sequencing.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Baym
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kryazhimskiy
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Tami D
authors.initials: TD
authors.lastname: Lieberman
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Hattie
authors.initials: H
authors.lastname: Chung
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America; FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America; Department of Physics, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Desai
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America; Faculty of Biology and Department of Computer Science, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Roy
authors.initials: R
authors.lastname: Kishony
mesh_terms: D015723:Gene Library; D023281:Genomics / Q000191:economics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods