Next Generation DNA sequencing (NGS) offers many potential benefits, but major factors limiting NGS include reducing the time and costs associated with: 1) start-up (i.
More...Next Generation DNA sequencing (NGS) offers many potential 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 workers have focused on reducing sample preparation costs, few workers 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 (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 (99.0% success). We also tested the indexed primers in real-world library preparation 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 different researchers to use their own primer sets which facilitates pooling of samples, during sequencing, among projects and researchers. 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.
Less...Accession | PRJNA321721 |
Data Type | Raw sequence reads |
Scope | Multispecies |
Grants | - "Dimensions: Testing the Potential of Pathogenic Fungi to Control the Diversity, Distribution, and Abundance of Tree Species in a Neotropical Forest Community" (Grant ID DEB-1136626, National Science Foundation)
- "Collaborative research: Remote sensing of foliar chemistry to reconstruct the genealogy of canopy trees in a Neotropical rain forest." (Grant ID DEB-1146440, National Science Foundation)
- "Collaborative Research: EAGER: Using ultraconserved elements (UCEs) as genomic markers to study shallow levels of evolutionary divergence" (Grant ID DEB-1242241, National Science Foundation)
- "Collaborative Research: EAGER: Using ultraconserved elements (UCEs) as genomic markers to study shallow levels of evolutionary divergence" (Grant ID DEB-1242260, National Science Foundation)
- "PIRE: Genetics of Invasive Species Exchanged Between the Southeastern U.S. and China, Taiwan and Hong Kong" (Grant ID OISE-0730218, National Science Foundation)
|
Submission | Registration date: 16-May-2016 Louisiana State University |
Related Resources | |
Relevance | Evolution |
Project Data:
No public data is linked to this project. Any recently released data that cites this project will be linked to it within a few days.