Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that 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. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

47Next-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 58All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/049114 doi: bioRxiv preprint first posted online Jun. 15, 2016; 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 632) 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

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Keywords: 3RAD, dual digest RADseq, ddRAD, DNA libraries, multiplexing, NGS. Bldg., University of Georgia, Athens, GA 30602, USA; 3 2 E-mail: njbayona@uga.edu . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/205799 doi: bioRxiv preprint first posted online Oct. 19, 2017; 2 Running Title: 3RAD -triple-enzyme RADseq digestion of genomic DNA to sample consistent portions of the genome from individuals being 4 0 studied (e.g., RADseq, GBS). However, researchers often find the existing methods expensive 4 1 to initiate and/or difficult to implement consistently. Here, we present a low-cost and highly integrating adapter designs that can be used interchangeably; 6) incorporating variable-length 4 9 internal tags within the adapters to increase the scope of sample tagging and facilitate pooling 5 0 while also increasing sequence diversity; 7) maintaining compatibility with universal dual- discover thousands of variable loci in each species.