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

Pacific trout Oncorhynchus spp. in western North America are strongly valued in ecological, socioeconomic, and cultural views, and have been the subject of substantial research and conservation efforts. Despite this, the understanding of their evolutionary histories, overall diversity, and challenges to their conservation is incomplete. We review the state of knowledge on these important issues, focusing on Pacific trout in the genus Oncorhynchus. Although most research on salmonid fishes emphasizes Pacific salmon, we focus on Pacific trout because they share a common evolutionary history, and many taxa in western North America have not been formally described, particularly in the southern extent of their ranges. Research in recent decades has led to the revision of many hypotheses concerning the origin and diversification of Pacific trout throughout their range. Although there has been significant success at addressing past threats to Pacific trout, contemporary and future threats represented by nonnative species, land and water use activities, and climate change pose challenges and uncertainties. Ultimately, conservation of Pacific trout depends on how well these issues are understood and addressed, and on solutions that allow these species to coexist with a growing scope of human influences. Conservación de la diversidad de truchas nativas del Pacífico en el oeste de NorteaméricaLa trucha del Pacífico Oncorhynchus spp. en el oeste de Norteamérica tiene un alto valor desde el punto de vista ecológi-co, socioeconómico y cultural, y ha sido objeto de importantes esfuerzos de conservación e investigación. A pesar de ello, el conocimiento que se tiene sobre su historia evolutiva, diversidad general y retos de conservación sigue siendo incompleto. Se hace una revisión del estado del conocimiento sobre estos puntos, con énfasis en la trucha del Pacífico dentro del género Oncorhynchus. Si bien la mayor parte de los estudios hechos sobre salmónidos se enfocan al salmón del Pací-fico, aquí nos enfocamos en la trucha del Pacífico ya que ambos groupos de especies comparten una historia evolutiva en común sobre todo en lo que se refiere al extremo sur de sus rangos de distribución. En investigaciones llevadas a cabo en décadas recientes, se han revisado varias hipótesis relativas al origen y diversificación de la trucha del Pacífico a lo largo de su rango de distribución. Aunque se han logrado identificar adecuadamente las amenazas pasadas que enfrentó la trucha del Pacífico, las amenazas actuales y futuras que representan especies no nativas, actividades de uso de tierra y agua y el cambio climático se consideran importantes retos e incertidumbres. Al final, la conservación de la trucha del Pacífico depende de qué tan bien se comprendan y abordan estos temas, y de las soluciones que les permitan a estas especies coexistir con una gama creciente de influencias humanas. Conservation de la diversité de la truite du Pacifique indigène dans l'ouest de l'Amérique du NordLes truites du Pacifique ou Oncorhynchus spp. dans l'ouest...

Aim:We explored the factors between riverscape features and exotic introgression of cultured rainbow trout (Oncorhynchus mykiss) that drive the current genetic structure in the endemic Mexican golden trout (Oncorhynchus chrysogaster) using a riverscape genetics simulator.Location: Sierra Madre Occidental, Northwest Mexico, North America. Methods:We created a system model for Mexican golden trout to test various scenarios for the effect of riverscape resistance and rainbow trout introgression on the native genetic structure of the Mexican golden trout. Individual genotypes were used to initialize the model and were based upon empirical allele frequencies from three genetically and spatially distinct groups in the study area. The geographical location of the simulated populations was defined using a species distribution model. We included hypotheses for movement of riverine distance and riverscape resistance. We evaluated model performance by conducting population and landscape genetics analyses on the simulation outputs. Results:The most plausible scenario included riverscape resistance without introgression, which presented a spatial genetic structure matching the observed riverscape genetic structure and fitting expected general life-history patterns for high elevation trout streams. In addition, temperature, precipitation, hydrology and topography were the most important variables influencing species occurrence and dispersal. Main conclusion:This work presents one of the first empirically derived simulation studies of introgression. It highlights the importance of considering riverscape resistance (i.e. hydroclimatic and topographical gradient effects) in models of spatial genetic structure and introgression. Moreover, our modelling suggests that what we characterize as riverscape resistance is acting as a boundary against exotic introgression. The results of this study could help to develop management strategies that preserve native gene pools.

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