Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

Promises and pitfalls of using high‐throughput sequencing for diet analysis

The application of high‐throughput sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high‐throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.     

Genotyping‐by‐sequencing approaches to characterize crop genomes: choosing the right tool for the right application

In the last decade, the revolution in sequencing technologies has deeply impacted crop genotyping practice. New methods allowing rapid, high‐throughput genotyping of entire crop populations have proliferated and opened the door to wider use of molecular tools in plant breeding. These new genotyping‐by‐sequencing (GBS) methods include over a dozen reduced‐representation sequencing (RRS) approaches and at least four whole‐genome resequencing (WGR) approaches. The diversity of methods available, each often producing different types of data at different cost, can make selection of the best‐suited method seem a daunting task. We review the most common genotyping methods used today and compare their suitability for linkage mapping, genomewide association studies (GWAS), marker‐assisted and genomic selection and genome assembly and improvement in crops with various genome sizes and complexity. Furthermore, we give an outline of bioinformatics tools for analysis of genotyping data. WGR is well suited to genotyping biparental cross populations with complex, small‐ to moderate‐sized genomes and provides the lowest cost per marker data point. RRS approaches differ in their suitability for various tasks, but demonstrate similar costs per marker data point. These approaches are generally better suited for de novo applications and more cost‐effective when genotyping populations with large genomes or high heterozygosity. We expect that although RRS approaches will remain the most cost‐effective for some time, WGR will become more widespread for crop genotyping as sequencing costs continue to decrease.     

CHIIMP: An automated high‐throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees

Short tandem repeats (STRs), also known as microsatellites, are commonly used to noninvasively genotype wild‐living endangered species, including African apes. Until recently, capillary electrophoresis has been the method of choice to determine the length of polymorphic STR loci. However, this technique is labor intensive, difficult to compare across platforms, and notoriously imprecise. Here we developed a MiSeq‐based approach and tested its performance using previously genotyped fecal samples from long‐term studied chimpanzees in Gombe National Park, Tanzania. Using data from eight microsatellite loci as a reference, we designed a bioinformatics platform that converts raw MiSeq reads into locus‐specific files and automatically calls alleles after filtering stutter sequences and other PCR artifacts. Applying this method to the entire Gombe population, we confirmed previously reported genotypes, but also identified 31 new alleles that had been missed due to sequence differences and size homoplasy. The new genotypes, which increased the allelic diversity and heterozygosity in Gombe by 61% and 8%, respectively, were validated by replicate amplification and pedigree analyses. This demonstrated inheritance and resolved one case of an ambiguous paternity. Using both singleplex and multiplex locus amplification, we also genotyped fecal samples from chimpanzees in the Greater Mahale Ecosystem in Tanzania, demonstrating the utility of the MiSeq‐based approach for genotyping nonhabituated populations and performing comparative analyses across field sites. The new automated high‐throughput analysis platform (available at https://github.com/ShawHahnLab/chiimp ) will allow biologists to more accurately and effectively determine wildlife population size and structure, and thus obtain information critical for conservation efforts.     

metaBIT,an integrative and automated metagenomic pipeline for analysing microbial profiles from high‐throughput sequencing shotgun data

Micro‐organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high‐throughput DNA sequencing (HTS). Here, we develop metaBIT, an open‐source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy‐based assignment and relative abundance calculation of microbial taxa, as well as cross‐sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present the diversity of outputs provided by the pipeline for the visualization of microbial profiles (barplots, heatmaps) and for their characterization and comparison (diversity indices, hierarchical clustering and principal coordinates analyses). We show that metaBIT allows an automatic, fast and user‐friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

Characterization of a Wheat Breeders’ Array suitable for high‐throughput SNP genotyping of global accessions of hexaploid bread wheat (Triticum aestivum)

Targeted selection and inbreeding have resulted in a lack of genetic diversity in elite hexaploid bread wheat accessions. Reduced diversity can be a limiting factor in the breeding of high yielding varieties and crucially can mean reduced resilience in the face of changing climate and resource pressures. Recent technological advances have enabled the development of molecular markers for use in the assessment and utilization of genetic diversity in hexaploid wheat. Starting with a large collection of 819 571 previously characterized wheat markers, here we describe the identification of 35 143 single nucleotide polymorphism‐based markers, which are highly suited to the genotyping of elite hexaploid wheat accessions. To assess their suitability, the markers have been validated using a commercial high‐density Affymetrix Axiom^® genotyping array (the Wheat Breeders’ Array), in a high‐throughput 384 microplate configuration, to characterize a diverse global collection of wheat accessions including landraces and elite lines derived from commercial breeding communities. We demonstrate that the Wheat Breeders’ Array is also suitable for generating high‐density genetic maps of previously uncharacterized populations and for characterizing novel genetic diversity produced by mutagenesis. To facilitate the use of the array by the wheat community, the markers, the associated sequence and the genotype information have been made available through the interactive web site ‘CerealsDB’.     

Thirty‐eight single nucleotide polymorphism markers for high‐throughput genotyping of chum salmon

We characterize 38 single nucleotide polymorphism genotyping assays for chum salmon (Oncorhynchus keta), an important species for both commercial and subsistence fisheries in western Alaska. These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Minor allele frequency differences (Δq) among collections were between 0.01 and 0.50 resulting in per locus F_ST estimates of 0.00–0.08 with an average of 0.03.     

Thirty‐two single nucleotide polymorphism markers for high‐throughput genotyping of sockeye salmon

We characterize 32 single nucleotide polymorphism genotyping assays for resolving genotypic variation in sockeye salmon Oncorhynchus nerka in the Pacific Rim. These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Minor allele frequency differences (Δq) among collections were between 4.7% and 97.9%, resulting in per locus F_ST estimates of 0.02–0.71 with an average of 0.22.     

High‐throughput genotyping for species identification and diversity assessment in germplasm collections

Germplasm collections provide an extremely valuable resource for breeders and researchers. However, misclassification of accessions by species often hinders the effective use of these collections. We propose that use of high‐throughput genotyping tools can provide a fast, efficient and cost‐effective way of confirming species in germplasm collections, as well as providing valuable genetic diversity data. We genotyped 180 Brassicaceae samples sourced from the Australian Grains Genebank across the recently released Illumina Infinium Brassica 60K SNP array. Of these, 76 were provided on the basis of suspected misclassification and another 104 were sourced independently from the germplasm collection. Presence of the A‐ and C‐genomes combined with principle components analysis clearly separated Brassica rapa, B. oleracea, B. napus, B. carinata and B. juncea samples into distinct species groups. Several lines were further validated using chromosome counts. Overall, 18% of samples (32/180) were misclassified on the basis of species. Within these 180 samples, 23/76 (30%) supplied on the basis of suspected misclassification were misclassified, and 9/105 (9%) of the samples randomly sourced from the Australian Grains Genebank were misclassified. Surprisingly, several individuals were also found to be the product of interspecific hybridization events. The SNP (single nucleotide polymorphism) array proved effective at confirming species, and provided useful information related to genetic diversity. As similar genomic resources become available for different crops, high‐throughput molecular genotyping will offer an efficient and cost‐effective method to screen germplasm collections worldwide, facilitating more effective use of these valuable resources by breeders and researchers.     

Oufti: an integrated software package for high‐accuracy,high‐throughput quantitative microscopy analysis

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.     

High‐throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high‐throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole‐genome amplification (WGA) by primer‐extension preamplification. We used the Agena Biosciences MassARRAY^® platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high‐throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.     

High‐throughput SNP discovery in the rabbit (Oryctolagus cuniculus) genome by next‐generation semiconductor‐based sequencing

The European rabbit (Oryctolagus cuniculus) is a domesticated species with one of the broadest ranges of economic and scientific applications and fields of investigation. Rabbit genome information and assembly are available (oryCun2.0), but so far few studies have investigated its variability, and massive discovery of polymorphisms has not been published yet for this species. Here, we sequenced two reduced representation libraries (RRLs) to identify single nucleotide polymorphisms (SNPs) in the rabbit genome. Genomic DNA of 10 rabbits belonging to different breeds was pooled and digested with two restriction enzymes (HaeIII and RsaI) to create two RRLs which were sequenced using the Ion Torrent Personal Genome Machine. The two RRLs produced 2 917 879 and 4 046 871 reads, for a total of 280.51 Mb (248.49 Mb with quality >20) and 417.28 Mb (360.89 Mb with quality >20) respectively of sequenced DNA. About 90% and 91% respectively of the obtained reads were mapped on the rabbit genome, covering a total of 15.82% of the oryCun2.0 genome version. The mapping and ad hoc filtering procedures allowed to reliably call 62 491 SNPs. SNPs in a few genomic regions were validated by Sanger sequencing. The Variant Effect Predictor Web tool was used to map SNPs on the current version of the rabbit genome. The obtained results will be useful for many applied and basic research programs for this species and will contribute to the development of cost‐effective solutions for high‐throughput SNP genotyping in the rabbit.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

Estimating and accounting for genotyping errors in RAD‐seq experiments

In non‐model organisms, evolutionary questions are frequently addressed using reduced representation sequencing techniques due to their low cost, ease of use, and because they do not require genomic resources such as a reference genome. However, evidence is accumulating that such techniques may be affected by specific biases, questioning the accuracy of obtained genotypes, and as a consequence, their usefulness in evolutionary studies. Here, we introduce three strategies to estimate genotyping error rates from such data: through the comparison to high quality genotypes obtained with a different technique, from individual replicates, or from a population sample when assuming Hardy‐Weinberg equilibrium. Applying these strategies to data obtained with Restriction site Associated DNA sequencing (RAD‐seq), arguably the most popular reduced representation sequencing technique, revealed per‐allele genotyping error rates that were much higher than sequencing error rates, particularly at heterozygous sites that were wrongly inferred as homozygous. As we exemplify through the inference of genome‐wide and local ancestry of well characterized hybrids of two Eurasian poplar (Populus) species, such high error rates may lead to wrong biological conclusions. By properly accounting for these error rates in downstream analyses, either by incorporating genotyping errors directly or by recalibrating genotype likelihoods, we were nevertheless able to use the RAD‐seq data to support biologically meaningful and robust inferences of ancestry among Populus hybrids. Based on these findings, we strongly recommend carefully assessing genotyping error rates in reduced representation sequencing experiments, and to properly account for these in downstream analyses, for instance using the tools presented here.     

Indel arrays: an affordable alternative for genotyping

Natural variation and induced mutations are important resources for gene discovery and the elucidation of genetic circuits. Mapping such polymorphisms requires rapid and cost-efficient methods for genome-wide genotyping. Here we report the development of a microarray-based method that assesses 240 unique markers in a single hybridization experiment at a cost of less than US$50 in materials per line. Our genotyping array is built with 70-mer oligonucleotide elements representing insertion/deletion (indel) polymorphisms between the Arabidopsis thaliana accessions Columbia-0 (Col) and Landsberg erecta (Ler). These indel polymorphisms are recognized with great precision by comparative genomic hybridization, eliminating the need for array replicates and complex statistical analysis. Markers are present genome-wide, with an average spacing of approximately 500 kb. PCR primer information is provided for all array indels, allowing rapid single-locus inquiries. Multi-well chips allow groups of 16 lines to be genotyped in a single experiment. We demonstrate the utility of the array for accurately mapping recessive mutations, RIL populations and mixed genetic backgrounds from accessions other than Col and Ler. Given the ease of use of shotgun sequencing to generate partial genomic sequences of unsequenced species, this approach is readily transferable to non-model organisms.     

SNP discovery and genotyping using restriction‐site‐associated DNA sequencing in chickens

Single nucleotide polymorphisms (SNPs) are essential to the understanding of population genetic variation and diversity. Here, we performed restriction‐site‐associated DNA sequencing (RAD‐seq) on 72 individuals from 13 Chinese indigenous and three introduced chicken breeds. A total of 620 million reads were obtained using an Illumina Hiseq2000 sequencer. An average of 75 587 SNPs were identified from each individual. Further filtering strictly validated 28 895 SNPs candidates for all populations. When compared with the NCBI dbSNP (chicken_9031), 15 404 SNPs were new discoveries. In this study, RAD‐seq was performed for the first time on chickens, implicating the remarkable effectiveness and potential applications on genetic analysis and breeding technique for whole‐genome selection in chicken and other agricultural animals.     

Unraveling hierarchical genetic structure in a marine metapopulation: A comparison of three high‐throughput genotyping approaches

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high‐throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high‐resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high‐throughput genotyping data can elucidate subtle genetic structure at previously‐undetected scales in a dispersive marine fish.     

Miniaturized fluid array for high‐throughput protein expression

We describe a miniaturized fluid array device for high‐throughput cell‐free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost‐prohibitive to implement in a high‐throughput format. As more and more new genes are being identified, there is a considerable need to have high‐throughput methods to produce a large number of proteins for studying structures and functions of the corresponding genes. The device consists of 96 units and each unit is for expression of one protein; thus up to 96 proteins can be produced simultaneously. The function of the fluid array was demonstrated by expression of a variety of proteins, with more than two orders of magnitude reduction in reagent consumption compared with a commercially available CFPS instrument. The protein expression yield in the device was up to 87 times higher for β‐glucoronidase than that in a conventional microplate. The concentration of β‐galactosidase expressed in the device was determined at 5.5 μg/μL. The feasibility of using the device for drug screening was demonstrated by measuring the inhibitory effects of mock drug compounds on synthesized β‐lactamase without the need for harvesting proteins, which enabled us to reduce the analysis time from days to hours. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010