Faculty, Scientist and Director
Department of Plant Biology
2502 Miller Plant Sciences
University of Georgia
Athens, GA 30602
Georgia Genomics and Bioinformatics Lab
110 Riverbend Rd., Room 161
Athens, GA 30602
Glenn, T., Nilsen, R., Kieran, T. J., Finger, J. W., Pierson, T. W., Bentley, K., … Faircloth, B. (2016). Universal stubs and primers for thousands of dual-indexed Illumina libraries 1 ( iTru & iNext ) 2 3.
Glenn, T., R. Nilsen, Troy J. Kieran, J. W. Finger, Todd W. Pierson, K. Bentley, Sandra L. Hoffberg, et al. “Universal Stubs and Primers for Thousands of Dual-Indexed Illumina Libraries 1 ( ITru & INext ) 2 3” (2016).
Glenn, T., et al. Universal Stubs and Primers for Thousands of Dual-Indexed Illumina Libraries 1 ( ITru & INext ) 2 3. 2016.
47 Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS 48 include reducing the time and costs associated with: 1) start-up (i.e., doing NGS for the first 49 time), 2) buy-in (i.e., getting any data from a run), and 3) sample preparation. Although many 50 researchers have focused on reducing sample preparation costs, few have addressed the first two 51 problems. Here, we present iTru and iNext, dual-indexing systems for Illumina libraries that 52 help address all three of these issues. By breaking the library construction process into re-usable, 53 combinatorial components, we achieve low start-up, buy-in, and per-sample costs, while 54 simultaneously increasing the number of samples that can be combined within a single run. We 55 accomplish this by extending the Illumina TruSeq dual-indexing approach from 20 (8+12) 56 indexed adapters that produce 96 (8x12) unique combinations to 579 (192+387) indexed primers 57 that produce 74,304 (192x387) unique combinations. We synthesized 208 of these indexed 58 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. 3 primers for validation, and 206 of them passed our validation criteria (99% success). We also 59 used the indexed primers to create hundreds of libraries in a variety of scenarios. Our approach 60 reduces start-up and per-sample costs by requiring only one universal adapter which works with 61 indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: 62 1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and 63 2) the large number of possible primers allows researchers to use unique primer sets for different 64 projects, which facilitates pooling of samples during sequencing. Although the methods we 65 present are highly customizable, resulting libraries can be used with the standard Illumina 66 sequencing primers and demultiplexed with the standard Illumina software packages, thereby 67 minimizing instrument and software customization headaches. In subsequent Adapterama 68 papers, we use these same iTru primers with different adapter stubs to construct double-to 69 quadruple-indexed amplicon libraries and double-digest restriction-site associated DNA (RAD) 70 libraries. For additional details and updates, please see http://baddna.org.