Adapterama I: Universal stubs and primers for thousands of dual-indexed Illumina libraries (iTru & iNext)

Abstract

Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing the time and costs associated with: 1) start-up (i.e., doing NGS for the first time), 2) buy-in (i.e., getting any data from a run), and 3) sample preparation. Although many researchers have focused on reducing sample preparation costs, few have addressed the first two problems. Here, we present iTru and iNext, dual-indexing systems for Illumina libraries that help address all three of these issues. By breaking the library construction process into re-usable, combinatorial components, we achieve low start-up, buy-in, and per-sample costs, while simultaneously increasing the number of samples that can be combined within a single run. We accomplish this by extending the Illumina TruSeq dual-indexing approach from 20 (8+12) indexed adapters that produce 96 (8x12) unique combinations to 579 (192+387) indexed primers that produce 74,304 (192x387) unique combinations. We synthesized 208 of these indexed primers for validation, and 206 of them passed our validation criteria (99% success). We also used the indexed primers to create hundreds of libraries in a variety of scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter which works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: 1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and 2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Although the methods we present are highly customizable, resulting libraries can be used with the standard Illumina sequencing primers and demultiplexed with the standard Illumina software packages, thereby minimizing instrument and software customization headaches. In subsequent Adapterama papers, we use these same iTru primers with different adapter stubs to construct double- to quadruple-indexed amplicon libraries and double-digest restriction-site associated DNA (RAD) libraries. For additional details and updates, please see http://baddna.org.