Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan TM genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GTseq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.

Unlike most anadromous fishes that have evolved strict homing behaviour, Pacific lamprey (Entosphenus tridentatus) seem to lack philopatry as evidenced by minimal population structure across the species range. Yet unexplained findings of within-region population genetic heterogeneity coupled with the morphological and behavioural diversity described for the species suggest that adaptive genetic variation underlying fitness traits may be responsible. We employed restriction site-associated DNA sequencing to genotype 4439 quality filtered single nucleotide polymorphism (SNP) loci for 518 individuals collected across a broad geographical area including British Columbia, Washington, Oregon and California. A subset of putatively neutral markers (N = 4068) identified a significant amount of variation among three broad populations: northern British Columbia, Columbia River/southern coast and 'dwarf' adults (F(CT) = 0.02, P ≪ 0.001). Additionally, 162 SNPs were identified as adaptive through outlier tests, and inclusion of these markers revealed a signal of adaptive variation related to geography and life history. The majority of the 162 adaptive SNPs were not independent and formed four groups of linked loci. Analyses with matsam software found that 42 of these outlier SNPs were significantly associated with geography, run timing and dwarf life history, and 27 of these 42 SNPs aligned with known genes or highly conserved genomic regions using the genome browser available for sea lamprey. This study provides both neutral and adaptive context for observed genetic divergence among collections and thus reconciles previous findings of population genetic heterogeneity within a species that displays extensive gene flow.

To elucidate the mechanisms of thermal adaptation and acclimation in ectothermic aquatic organisms from differing climates, we used a common-garden experiment for thermal stress to investigate the heat shock response of redband trout (Oncorhynchus mykiss gairdneri) from desert and montane populations. Evidence for adaptation was observed as expression of heat shock genes in fish from the desert population was more similar to control (unstressed) fish and significantly different (P ≤ 0.05) from those from the montane population, while F1 crosses were intermediate. High induction of heat shock proteins (Hsps) in the montane strain appeared to improve short-term survival during first exposure to high water temperatures, but high physiological costs of Hsp production may have led to lower long-term survival. In contrast, the desert strain had significantly lower heat shock response than the montane fish and F1 crosses, suggesting that these desert fish have evolved alternative mechanisms to deal with thermal stress that provide better balance of physiological costs. Genomewide tests of greater than 10 000 SNPs found multiple SNPs that were significantly associated with survival under thermal stress, including Hsp47 which consistently appeared as a strong candidate gene for adaption to desert climates. Candidate SNPs identified in this study are prime targets to screen more broadly across this species' range to predict the potential for adaptation under scenarios of climate change. These results demonstrate that aquatic species can evolve adaptive responses to thermal stress and provide insight for understanding how climate change may impact ectotherms.

Little is known of the genetic basis of migration despite the ecological benefits migratory species provide to their communities and their rapid global decline due to anthropogenic disturbances in recent years. Using next generation sequencing of restriction-site associated DNA (RAD) tags we genotyped thousands of SNPs in two wild populations of migratory steelhead and resident rainbow trout (Oncorhynchus mykiss) from the Pacific Northwest of the United States. One population maintains a connection to the sea, whereas the other population has been sequestered from its access to the ocean for more than fifty years by a hydropower dam. Here we performed a genome-wide association study (GWAS) to identify 504 RAD SNP markers from several genetic regions that were associated with the propensity to migrate both within and between the populations. Our results corroborate those in previous quantitative trait loci (QTL) studies and provide evidence for additional loci associated with this complex migratory life history. Our results suggest a complex multi-genic basis with several loci of small effect distributed throughout the genome contributing to migration in this species. We also determined that despite being sequestered for decades, the landlocked population continues to harbor genetic variation associated with a migratory life history and ATPase activity. Furthermore we demonstrate the utility of genotyping-by-sequencing and how RAD-tag SNP data can be readily compared between studies to investigate migration within this species.

Parentage-based tagging (PBT) is a promising alternative to traditional coded-wire tag (CWT) methodologies for monitoring and evaluating hatchery stocks. This approach involves the genotyping of hatchery broodstock and uses parentage assignments to identify the origin and brood year of their progeny. In this study we empirically confirmed that fewer than 100 single nucleotide polymorphisms (SNPs) were needed to accurately conduct PBT, we demonstrated that our selected panel of SNPs was comparable in accuracy to a panel of microsatellites, and we verified that stock assignments made with this panel matched those made using CWTs. We also demonstrated that when sampling of spawners was incomplete, an estimated PBT rate for the offspring could also be predicted with fewer than 100 SNPs. This study in the Snake River basin is one of the first large-scale implementations of PBT in salmonids and lays the foundation for adopting this technology more broadly in the region, thereby allowing the unprecedented ability to mark millions of smolts and an opportunity to address a variety of parentage-based research and management questions.

BackgroundAs ectothermic organisms have evolved to differing aquatic climates, the molecular basis of thermal adaptation is a key area of research. In this study, we tested for differential transcriptional response of ecologically divergent populations of redband trout (Oncorhynchus mykiss gairdneri) that have evolved in desert and montane climates. Each pure strain and their F1 cross were reared in a common garden environment and exposed over four weeks to diel water temperatures that were similar to those experienced in desert climates within the species’ range. Gill tissues were collected from the three strains of fish (desert, montane, F1 crosses) at the peak of heat stress and tested for mRNA expression differences across the transcriptome with RNA-seq.ResultsStrong differences in transcriptomic response to heat stress were observed across strains confirming that fish from desert environments have evolved diverse mechanisms to cope with stressful environments. As expected, a large number of total transcripts (12,814) were differentially expressed in the study (FDR ≤ 0.05) with 2310 transcripts in common for all three strains, but the desert strain had a larger number of unique differentially expressed transcripts (2875) than the montane (1982) or the F1 (2355) strain. Strongly differentiated genes (>4 fold change and FDR ≤ 0.05) were particularly abundant in the desert strain (824 unique contigs) relative to the other two strains (montane = 58; F1 = 192).ConclusionsThis study demonstrated patterns of acclimation (i.e., phenotypic plasticity) within strains and evolutionary adaptation among strains in numerous genes throughout the transcriptome. Key stress response genes such as molecular chaperones (i.e., heat shock proteins) had adaptive patterns of gene expression among strains, but also a much higher number of metabolic and cellular process genes were differentially expressed in the desert strain demonstrating these biological pathways are critical for thermal adaptation to warm aquatic climates. The results of this study further elucidate the molecular basis for thermal adaptation in aquatic ecosystems and extend the potential for identifying genes that may be critical for adaptation to changing climates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1246-5) contains supplementary material, which is available to authorized users.

Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.