Troy J. Kieran

Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double‐digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

RADcap: sequence capture of dual-digest RADseq libraries with identifiable duplicates and reduced missing data.

Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods, such as restriction‐site‐associated DNA sequencing (RADseq) and sequence capture, are constrained by costs associated with inefficient use of sequencing data and sample preparation. Here, we introduce RADcap, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches. RADcap uses a new version of dual‐digest RADseq (3RAD) to identify candidate SNP loci for capture bait design and subsequently uses custom sequence capture baits to consistently enrich candidate SNP loci across many individuals. We combined this approach with a new library preparation method for identifying and removing PCR duplicates from 3RAD libraries, which allows researchers to process RADseq data using traditional pipelines, and we tested the RADcap method by genotyping sets of 96–384 Wisteria plants. Our results demonstrate that our RADcap method: (i) methodologically reduces (to <5%) and allows computational removal of PCR duplicate reads from data, (ii) achieves 80–90% reads on target in 11 of 12 enrichments, (iii) returns consistent coverage (≥4×) across >90% of individuals at up to 99.8% of the targeted loci, (iv) produces consistently high occupancy matrices of genotypes across hundreds of individuals and (v) costs significantly less than current approaches.

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.Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing costs of: 1) start-up (i.e., doing NGS for the first time); 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.

Conflicting Evolutionary Histories of the Mitochondrial and Nuclear Genomes in New World Myotis Bats

&NA; The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re‐examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories.

The Novel Evolution of the Sperm Whale Genome

Abstract The sperm whale, made famous by Moby Dick, is one of the most fascinating of all ocean-dwelling species given their unique life history, novel physiological adaptations to hunting squid at extreme ocean depths, and their position as one of the earliest branching toothed whales (Odontoceti). We assembled the sperm whale (Physeter macrocephalus) genome and resequenced individuals from multiple ocean basins to identify new candidate genes for adaptation to an aquatic environment and infer demographic history. Genes crucial for skin integrity appeared to be particularly important in both the sperm whale and other cetaceans. We also find sperm whales experienced a steep population decline during the early Pleistocene epoch. These genomic data add new comparative insight into the evolution of whales.

Adapterama IV: Sequence Capture of Dual-digest RADseq Libraries with Identifiable Duplicates (RADcap)

Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods such as restriction site associated DNA sequencing (RADseq) and sequence capture are constrained by costs associated with inefficient use of sequencing data and sample preparation, respectively. Here, we demonstrate RADcap, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches. The RADcap approach uses a new version of dual-digest RADseq (3RAD) to identify candidate SNP loci for capture bait design, and subsequently uses custom sequence capture baits to consistently enrich candidate SNP loci across many individuals. We combined this approach with a new library preparation method for identifying and removing PCR duplicates from 3RAD libraries, which allows researchers to process RADseq data using traditional pipelines, and we tested the RADcap method by genotyping sets of 96 to 384 Wisteria plants. Our results demonstrate that our RADcap method: 1) can methodologically reduce (to <5%) and computationally remove PCR duplicate reads from data; (2) achieves 80-90% reads-on-target in 11 of 12 enrichments; (3) returns consistent coverage (≥4x) across >90% of individuals at up to 99.9% of the targeted loci; (4) produces consistently high occupancy matrices of genotypes across hundreds of individuals; and (5) is inexpensive, with reagent and sequencing costs totaling <

Blood Meal Source Characterization Using Illumina Sequencing in the Chagas Disease Vector Rhodnius pallescens (Hemiptera: Reduviidae) in Panamá

6/sample and adapter and primer costs of only a few hundred dollars.

Insight from an Ultraconserved Element Bait Set Designed for Hemipteran Phylogenetics Integrated with Genomic Resources

Abstract Accurate blood meal identification is critical to understand hematophagous vector-host relationships. This study describes a customizable Next-Generation Sequencing (NGS) approach to identify blood meals from Rhodnius pallescens (Hemiptera: Reduviidae) triatomines using multiple barcoded primers and existing software to pick operational taxonomic units and match sequences for blood meal identification. We precisely identified all positive control samples using this method and further examined 74 wild-caught R. pallescens samples. With this novel blood meal identification method, we detected 13 vertebrate species in the blood meals, as well as single and multiple blood meals in individual bugs. Our results demonstrate the reliability and descriptive uses of our method.

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about

Target enrichment of conserved genomic regions facilitates collecting sequences of many orthologous loci from non-model organisms to address phylogenetic, phylogeographic, population genetic, and molecular evolution questions. Bait sets for sequence capture can simultaneously target thousands of loci, which opens new avenues of research on speciose groups. Current phylogenetic hypotheses on the >103,000 species of Hemiptera have failed to unambiguously resolve major nodes, suggesting that alternative datasets and more thorough taxon sampling may be required to resolve relationships. We use a recently designed ultraconserved element (UCE) bait set for Hemiptera, with a focus on the suborder Heteroptera, or the true bugs, to test previously proposed relationships. We present newly generated UCE data for 36 samples representing three suborders, all seven heteropteran infraorders, 23 families, and 34 genera of Hemiptera and one thysanopteran outgroup. To improve taxon sampling, we also mined additional UCE loci in silico from published hemipteran genomic and transcriptomic data. We obtained 2271 UCE loci for newly sequenced hemipteran taxa, ranging from 265 to 1696 (average 904) per sample. These were similar in number to the data mined from transcriptomes and genomes, but with fewer loci overall. The amount of missing data correlates with greater phylogenetic divergence from taxa used to design the baits. This bait set hybridizes to a wide range of hemipteran taxa and specimens of varying quality, including dried specimens as old as 1973. Our estimated phylogeny yielded topologies consistent with other studies for most nodes and was strongly-supported. We also demonstrate that UCE loci are almost exclusively from the transcribed portion of the genome, thus data can be successfully integrated with existing genomic and transcriptomic resources for more comprehensive phylogenetic sampling, an important feature in the era of phylogenomics. UCE approaches can be used by other researchers for additional studies on hemipteran evolution and other research that requires well resolved phylogenies.

1USD per Sample (3RAD)

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 due to the inability to highly-multiplex 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 relies 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.Molecular ecologists have used genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from individuals being studied (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently. Here, we present a low-cost and highly robust approach for the construction of dual-digest RADseq libraries. 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; 5) integrating adapter designs that can be used interchangeably; 6) incorporating variable-length internal tags within the adapters to increase the scope of sample tagging and facilitate pooling while also increasing sequence diversity; 7) maintaining compatibility with universal dual-indexed primers, which facilitate construction of combinatorial quadruple-indexed libraries that are compatible with standard Illumina sequencing reagents and libraries; and, 8) easy tuning for molecular tagging and PCR duplicate identification. We present eight adapter designs that work with 72 restriction enzyme combinations, and we demonstrate our approach by the use of one set of adapters and one set of restriction enzymes across a variety of non-model organisms to discover thousands of variable loci in each species.