Adapterama I: Universal Stubs and Primers for Thousands of Dual-Indexed Illumina Libraries (iTru & iNext)

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.

[1]  Michael E Alfaro,et al.  Replicated divergence in cichlid radiations mirrors a major vertebrate innovation , 2016, Proceedings of the Royal Society B: Biological Sciences.

[2]  Jonathan P. Bollback,et al.  The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing , 2007, PloS one.

[3]  S. Aljanabi,et al.  Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. , 1997, Nucleic acids research.

[4]  Martin Kircher,et al.  Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform , 2011, Nucleic acids research.

[5]  W. Ansorge.  Next-generation DNA sequencing techniques. , 2009, New biotechnology.

[6]  Andrew C. Adey,et al.  Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition , 2010, Genome Biology.

[7]  Pindaro Diaz-Jaimes,et al.  The complete mitochondrial DNA of white shark (Carcharodon carcharias) from Isla Guadalupe, Mexico , 2016, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[8]  Kurt D. Reed,et al.  Genetic diversity in Blastomyces dermatitidis: implications for PCR detection in clinical and environmental samples. , 2009 .

[9]  Matthew J. Huentelman,et al.  IDENTIFICATION OF GENETIC VARIANTS USING BARCODED MULTIPLEXED SEQUENCING , 2008, Nature Methods.

[10]  T. Glenn.  Field guide to next‐generation DNA sequencers , 2011, Molecular ecology resources.

[11]  B. Faircloth,et al.  Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels , 2012, PloS one.

[12]  Matt Friedman,et al.  Phylogenomic analysis of carangimorph fishes reveals flatfish asymmetry arose in a blink of the evolutionary eye , 2016, BMC Evolutionary Biology.

[13]  B. Faircloth,et al.  Target capture and massively parallel sequencing of ultraconserved elements for comparative studies at shallow evolutionary time scales. , 2013, Systematic biology.

[14]  Travis C Glenn,et al.  Ultraconserved elements anchor thousands of genetic markers spanning multiple evolutionary timescales. , 2012, Systematic biology.

[15]  Haiying Li Grunenwald,et al.  Next-generation sequencing library preparation: simultaneous fragmentation and tagging using in vitro transposition , 2009 .

[16]  Marina Marcet-Houben,et al.  The complete mitochondrial DNA of the silky shark (Carcharhinus falciformis) , 2016, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[17]  Seán G. Brady,et al.  Target enrichment of ultraconserved elements from arthropods provides a genomic perspective on relationships among Hymenoptera , 2014, Molecular ecology resources.

[18]  Travis C Glenn,et al.  RADcap: sequence capture of dual‐digest RADseq libraries with identifiable duplicates and reduced missing data , 2016, Molecular ecology resources.

[19]  Travis C. Glenn,et al.  A Phylogeny of Birds Based on Over 1,500 Loci Collected by Target Enrichment and High-Throughput Sequencing , 2012, PloS one.

[20]  F. Bushman,et al.  DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations , 2007, Nucleic acids research.

[21]  Matthias Meyer,et al.  Illumina sequencing library preparation for highly multiplexed target capture and sequencing. , 2010, Cold Spring Harbor protocols.

[22]  Z. Ning,et al.  Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of GC-biased genomes , 2009, Nature Methods.

[23]  Bonnie B. Blaimer,et al.  Phylogenomics, biogeography and diversification of obligate mealybug-tending ants in the genus Acropyga. , 2016, Molecular phylogenetics and evolution.

[24]  Dennis C. Friedrich,et al.  A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries , 2011, Genome Biology.

[25]  D. Reich,et al.  Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture , 2012, Genome research.

[26]  P. Díaz‐Jaimes,et al.  Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about $1USD per Sample (3RAD) , 2017, bioRxiv.

[27]  Detlef Weigel,et al.  Next Generation Molecular Ecology , 2010, Molecular ecology.

[28]  Ronald W. Davis,et al.  Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar–coding strategy , 1996, Nature Genetics.

[29]  Troy J. Kieran,et al.  Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq) , 2015, Molecular ecology resources.

[30]  E. Balart,et al.  The complete mitochondrial DNA of endemic Eastern Pacific coral (Porites panamensis) , 2016, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[31]  Nicholas G. Crawford,et al.  More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs , 2012, Biology Letters.

[32]  F. van Nieuwerburgh,et al.  Library construction for next-generation sequencing: overviews and challenges. , 2014, BioTechniques.

[33]  Nancy F. Hansen,et al.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry , 2008, Nature.

[34]  A Skerra,et al.  Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. , 1992, Nucleic acids research.

[35]  U. Stenzel,et al.  Targeted high-throughput sequencing of tagged nucleic acid samples , 2007, Nucleic acids research.

[36]  B. Faircloth,et al.  msatcommander: detection of microsatellite repeat arrays and automated, locus‐specific primer design , 2008, Molecular ecology resources.

[37]  T. Fennell,et al.  Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries , 2011, Genome Biology.

[38]  Todd A. Castoe,et al.  Rapid Microsatellite Identification from Illumina Paired-End Genomic Sequencing in Two Birds and a Snake , 2012, PloS one.