Targeted Sequence Capture Research Articles

Mutation analysis of VWF gene in two patients with von Willebrand disease by target sequence capture and high-throughput sequencing technology

Mutation analysis of VWF gene in two patients with von Willebrand disease by target sequence capture and high-throughput sequencing technology

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

Premise of the StudyTargeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single‐copy nuclear loci. Most published studies in plants have used taxon‐specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single‐copy and have orthologs in large clades of plants.MethodsWe identify and compare a taxon‐specific locus set and three general locus sets (conserved ortholog set [COSII], shared single‐copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees.ResultsThe taxon‐specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon‐specific and PPR loci had the highest average variability. The taxon‐specific data set produced the best‐supported tree, but all data sets showed improved resolution over previous non‐sequence capture data sets.DiscussionGeneral locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Exploring Approximate Bayesian Computation for inferring recent demographic history with genomic markers in nonmodel species.

Approximate Bayesian computation (ABC) is widely used to infer demographic history of populations and species using DNA markers. Genomic markers can now be developed for nonmodel species using reduced representation library (RRL) sequencing methods that select a fraction of the genome using targeted sequence capture or restriction enzymes (genotyping-by-sequencing, GBS). We explored the influence of marker number and length, knowledge of gametic phase, and tradeoffs between sample size and sequencing depth on the quality of demographic inferences performed with ABC. We focused on two-population models of recent spatial expansion with varying numbers of unknown parameters. Performing ABC on simulated data sets with known parameter values, we found that the timing of a recent spatial expansion event could be precisely estimated in a three-parameter model. Taking into account uncertainty in parameters such as initial population size and migration rate collectively decreased the precision of inferences dramatically. Phasing haplotypes did not improve results, regardless of sequence length. Numerous short sequences were as valuable as fewer, longer sequences, and performed best when a large sample size was sequenced at low individual depth, even when sequencing errors were added. ABC results were similar to results obtained with an alternative method based on the site frequency spectrum (SFS) when performed with unphased GBS-type markers. We conclude that unphased GBS-type data sets can be sufficient to precisely infer simple demographic models, and discuss possible improvements for the use of ABC with genomic data.

Filtering of target sequence capture individuals facilitates species tree construction in the plant subtribe Iochrominae (Solanaceae)

Advances in sequencing technology have made it possible to produce large multi-locus datasets required for species tree analyses. One challenge with constructing high throughput sequencing datasets, however, is that missing information is propagated at different steps in the sequence preparation process. To date, species tree studies have focused on filtering and removing errors that occur at particular loci. Given the way that high throughput sequencing datasets are constructed, however, large amounts of error or ambiguity may also manifest across individuals. Here we use a novel tree-based multivariate clustering method to identify and remove individuals with low phylogenetic signal in a nuclear sequence capture dataset for the Iochrominae clade (Solanaceae). Our results suggest that the low quality tips are the result of the library preparation process (e.g. unequal pooling) rather than poor capture due to phylogenetic distance from the reference species. After implementing the clustering approach and removing low quality tips, we construct an Iochrominae species tree that resolves a number of unknown relationships. We propose this pipeline as a valuable tool for species tree reconstruction with phylogenomic datasets containing variable levels of missing data.

In-depth resistome analysis by targeted metagenomics

BackgroundAntimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of “resistomes” (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes.MethodsWe developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect “gene abundance” (from 2.0 to 83.2%) and “gene diversity” (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof.ConclusionsResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes related to the selection and transfer of antimicrobial resistance (biocides, heavy metals, plasmids). The model opens the possibility to study other complex microbial systems in which minority populations play a relevant role.

Genetic Mapping and Phylogenetic Analysis Reveal Intraspecific Variation in Sex Chromosomes of the Virginian Strawberry.

With their extraordinary diversity in sexual systems, flowering plants offer unparalleled opportunities to understand sex determination and to reveal generalities in the evolution of sex chromosomes. Comparative genetic mapping of related taxa with good phylogenetic resolution can delineate the extent of sex chromosome diversity within plant groups, and lead the way to understanding the evolutionary drivers of such diversity. The North American octoploid wild strawberries provide such an opportunity. We performed linkage mapping using targeted sequence capture for the subdioecious western Fragaria virginiana ssp. platypetala and compared the location of its sex-determining region (SDR) to those of 2 other (sub)dioecious species, the eastern subspecies, F. virginiana ssp. virginiana (whose SDR is at 0-5.5 Mb on chromosome VI of the B2 subgenome), and the sister species F. chiloensis (whose SDR is at 37 Mb on chromosome VI of the Av subgenome). Male sterility was dominant in F. virginiana ssp. platypetala and mapped to a chromosome also in homeologous group VI. Likewise, one major quantitative trait locus (QTL) for female fertility overlapped the male sterility region. However, the SDR mapped to yet another subgenome (B1), and to a different location (13 Mb), but similar to the location inferred in one population of the naturally occurring hybrid between F. chiloensis and F. virginiana (F. ×ananassa ssp. cuneifolia). Phylogenetic analysis of chromosomes across the octoploid taxa showed consistent subgenomic composition reflecting shared evolutionary history but also reinforced within-species variation in the SDR-carrying chromosome, suggesting either repeated evolution, or recent turnovers in SDR.

Use of targeted sequence capture and high-throughput sequencing identifies a novel PKD1 mutation involved in adult polycystic kidney disease

Polycystic kidney disease (PKD) is a common inherited disease that is characterized by a progressive development of renal cysts. Approximately 85% of PKD cases are due to mutations in the polycystin 1 (PKD1) gene. Here, we report a pedigree containing nine patients with autosomal dominant PKD (ADPKD). Using targeted exome sequencing of PKD1 and PKD2 genes, we identified a novel heterozygous frameshift mutation c.3976_3977insCT (p.F1326Sfs*21) in the PKD1 gene that segregated between affected and unaffected family members. This mutation is currently not present in the 1000 Genomes Project nor ExAC databases and is therefore a novel PKD1 mutation involved in ADPKD. These results provide a novel sequence variant for the genetic analysis of this disease.

Phylogenomic analyses of more than 4000 nuclear loci resolve the origin of snakes among lizard families.

Squamate reptiles (lizards and snakes) are the most diverse group of terrestrial vertebrates, with more than 10 000 species. Despite considerable effort to resolve relationships among major squamates clades, some branches have remained difficult. Among the most vexing has been the placement of snakes among lizard families, with most studies yielding only weak support for the position of snakes. Furthermore, the placement of iguanian lizards has remained controversial. Here we used targeted sequence capture to obtain data from 4178 nuclear loci from ultraconserved elements from 32 squamate taxa (and five outgroups) including representatives of all major squamate groups. Using both concatenated and species-tree methods, we recover strong support for a sister relationship between iguanian and anguimorph lizards, with snakes strongly supported as the sister group of these two clades. These analyses strongly resolve the difficult placement of snakes within squamates and show overwhelming support for the contentious position of iguanians. More generally, we provide a strongly supported hypothesis of higher-level relationships in the most species-rich tetrapod clade using coalescent-based species-tree methods and approximately 100 times more loci than previous estimates.

P106 Whole gene, whole haplotype and whole genome sequencing in human

Aim Recent advances in sequencing, sample and library preparation technologies made it possible to sequence entire genes, haplotypes and genome in human. In this study, we used four approaches; amplicon based whole gene sequencing, capture probe based MHC haplotype sequencing, PacBio Sequel system based whole genome sequencing and whole genome mapping based on Bionanogenomics’ Technology to study one individual’s MHC. We wanted to compare the information that we have obtained from each technology and confirm the haplotype predictions. Methods We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al., Hum Immunol. 2015 Dec;76(12):963–74). We performed MHC region target sequence capture using Roche-NimbleGen SeqCap EZ Library. Whole genome sequencing We prepared 40 to 50 kb libraries with 15 to 20 kb cut off size selection and followed manufacturer’s protocol thereafter. Sequencing performed on PacBio Sequel® platform using v2 chemistry and v4 software. Movie time for these large fragments were set to 600 min and secondary analysis is done with SMRTLink’s (v4.0.0) SMRT Analysis Application: de novo Assembly (HGAP 4). To get 85× coverage, we have sequenced the same library in 62 different SMRT® Cell 1 M v2. Whole genome de novo assembly of PacBio sequencing is being performed by DNANexus. BioNano genome mapping was performed by Bio Nano genomics. Results Haplotype determination of the individual tested with whole gene amplicon sequencing performed based on the family studies and is being compared to haplotypes determined by other methods. Conclusions Now thanks to novel technologies we can decipher the complex genetic structure of MHC and other immune response genes with higher precision. This in turn will increase our ability to understand the genetic basis of the complex diseases.

Analysis of variants in complement genes in Han Chinese children with atypical hemolytic uremic syndrome

Objective: To investigate the prevalence and characteristics of pathogenic variants in complement genes in Han Chinese children with atypical hemolytic uremic syndrome (aHUS). Method: Eleven Han Chinese children with aHUS, including 9 boys and 2 girls aged between 1 year and 4 months and 13 years, were investigated in Department of Pediatrics, Fuzhou General Hospital, from November 1998 to February 2014. Analysis of variants of all the exons of 10 complement genes (CFH, MCP, CFI, C3, CFB, CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5), including 25 bases from 3' end and 25 bases from 5' end, was performed in the 11 cases by targeted sequence capture and next generation sequencing. Significant variants detected by next generation sequencing were confirmed by Sanger sequencing. To understand pathogenicity of variants found in the captured genes, we investigated genetic conservation by multiple protein sequence alignment among different species, and analyzed whether the variants were located in protein domains or not, and investigated functional significance by functional computational prediction methods. Result: Twenty-seven percent of Han Chinese children with aHUS carried pathogenic variants in the 10 complement genes. Pathogenic variant CFB 221G>A (R74H) was detected in Patient 3 and Patient 9, which was not found in parents of Patient 3' , and was found in healthy father of patient 9. Pathogenic variant CFHR5 242C>T (P81L) was found in Patient 2, and was found in healthy father of patient 2. However, no pathogenic variants in genes CFH, MCP, CFI, C3, CFHR1, CFHR2, CFHR3 and CFHR4 were identified. Conclusion: Pathogenic variants in the 10 complement genes were identified in 3/11 of Han Chinese children with aHUS in our study and CFB was the most frequently mutated gene.

Analysis of TCIRG1 gene mutation in a Chinese family affected with infantile malignant osteopetrosis

To detect potential mutation of the TCIRG1 gene in a boy with infantile malignant osteopetrosis. Target sequence capture and next-generation sequencing were applied for the proband and his parents to identify the causative mutation, and Sanger sequencing was used to verify the suspected mutation. The proband manifested at 4 months of age with symptoms including anemia, thrombocytopenia, hepatosplenomegaly, and cephalus quadratus. X-ray revealed generalized increased bone density. A novel compound heterozygous mutation, c.796G to T (p.E266X) and c.1372G to A (p.G458S), were identified in the boy. His father and grandmother also carried the c.796G to T (p.E266X) mutation, and his mother carried the c.1372G to A (p.G458S) mutation. Neither mutation was found in the PubMed and ClinVar databases. The novel compound heterozygous mutation c.796G to T (p.E266X) and c.1372G to A (p.G458S) probably underlies the disease in the proband. Above results may enrich the mutation spectrum of the TCIRG1 gene and provide new evidence for the molecular basis of infantile malignant osteopetrosis.

Identification of Single Nucleotide Polymorphism from Indian <i>Bubalus bubalis</i> through Targeted Sequence Capture

Identification of Single Nucleotide Polymorphism from Indian <i>Bubalus bubalis</i> through Targeted Sequence Capture

High-Throughput Sequencing of the Major Histocompatibility Complex following Targeted Sequence Capture.

The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using microarray-based genome-wide SNP analysis. Taken together, the use of recently developed tools and protocols for sequence capture and massively parallel sequencing allows for detailed MHC analysis and donor-recipient matching.

Expanding anchored hybrid enrichment to resolve both deep and shallow relationships within the spider tree of life.

BackgroundDespite considerable effort, progress in spider molecular systematics has lagged behind many other comparable arthropod groups, thereby hindering family-level resolution, classification, and testing of important macroevolutionary hypotheses. Recently, alternative targeted sequence capture techniques have provided molecular systematics a powerful tool for resolving relationships across the Tree of Life. One of these approaches, Anchored Hybrid Enrichment (AHE), is designed to recover hundreds of unique orthologous loci from across the genome, for resolving both shallow and deep-scale evolutionary relationships within non-model systems. Herein we present a modification of the AHE approach that expands its use for application in spiders, with a particular emphasis on the infraorder Mygalomorphae.ResultsOur aim was to design a set of probes that effectively capture loci informative at a diversity of phylogenetic timescales. Following identification of putative arthropod-wide loci, we utilized homologous transcriptome sequences from 17 species across all spiders to identify exon boundaries. Conserved regions with variable flanking regions were then sought across the tick genome, three published araneomorph spider genomes, and raw genomic reads of two mygalomorph taxa. Following development of the 585 target loci in the Spider Probe Kit, we applied AHE across three taxonomic depths to evaluate performance: deep-level spider family relationships (33 taxa, 327 loci); family and generic relationships within the mygalomorph family Euctenizidae (25 taxa, 403 loci); and species relationships in the North American tarantula genus Aphonopelma (83 taxa, 581 loci). At the deepest level, all three major spider lineages (the Mesothelae, Mygalomorphae, and Araneomorphae) were supported with high bootstrap support. Strong support was also found throughout the Euctenizidae, including generic relationships within the family and species relationships within the genus Aptostichus. As in the Euctenizidae, virtually identical topologies were inferred with high support throughout Aphonopelma.ConclusionsThe Spider Probe Kit, the first implementation of AHE methodology in Class Arachnida, holds great promise for gathering the types and quantities of molecular data needed to accelerate an understanding of the spider Tree of Life by providing a mechanism whereby different researchers can confidently and effectively use the same loci for independent projects, yet allowing synthesis of data across independent research groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0769-y) contains supplementary material, which is available to authorized users.

P5022 Identification of genetic markers associated with feeding efficiency in fattening Holstein calves, using targeted sequence capture

P5022 Identification of genetic markers associated with feeding efficiency in fattening Holstein calves, using targeted sequence capture

Identification of SLC26A4 mutations p.L582LfsX4, p.I188T and p.E704K in a Chinese family with large vestibular aqueduct syndrome (LVAS)

Large vestibular aqueduct syndrome (LVAS) is a type of hearing loss characterized by an autosomal recessive inheritance. LVAS has been shown to be associated with mutations in SLC26A4 gene. In the present study, we report the clinical, genetic and molecular characterization of a Chinese family with LVAS. By using the targeted sequence capture and next-generation sequencing, we identified heterozygous mutations of SLC26A4 p.I188T (c.563T > C), p.L582LfsX4 (c.1746 delG) and p.E704K (c.2110G > A) in the affected individual of this family, of which SLC26A4 p.E704K is a novel mutation associated with LVAS. By tracing the transmission and functional prediction of these mutations in the pedigree, the heterozygous mutations of p.I188T, p.L582LfsX4 and p.E704K in SLC26A4 gene were responsible for the LVAS of the affected individual. This is the first case of LVAS caused by these mutations.

Studying the genetic basis of speciation in high gene flow marine invertebrates.

A growing number of genes responsible for reproductive incompatibilities between species (barrier loci) exhibit the signals of positive selection. However, the possibility that genes experiencing positive selection diverge early in speciation and commonly cause reproductive incompatibilities has not been systematically investigated on a genome-wide scale. Here, I outline a research program for studying the genetic basis of speciation in broadcast spawning marine invertebrates that uses a priori genome-wide information on a large, unbiased sample of genes tested for positive selection. A targeted sequence capture approach is proposed that scores single-nucleotide polymorphisms (SNPs) in widely separated species populations at an early stage of allopatric divergence. The targeted capture of both coding and non-coding sequences enables SNPs to be characterized at known locations across the genome and at genes with known selective or neutral histories. The neutral coding and non-coding SNPs provide robust background distributions for identifying FST-outliers within genes that can, in principle, identify specific mutations experiencing diversifying selection. If natural hybridization occurs between species, the neutral coding and non-coding SNPs can provide a neutral admixture model for genomic clines analyses aimed at finding genes exhibiting strong blocks to introgression. Strongylocentrotid sea urchins are used as a model system to outline the approach but it can be used for any group that has a complete reference genome available.

A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44000year-old rodent remains from Northern Africa.

We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120000years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.

Genotyping by Sequencing Using Specific Allelic Capture to Build a High-Density Genetic Map of Durum Wheat.

Targeted sequence capture is a promising technology which helps reduce costs for sequencing and genotyping numerous genomic regions in large sets of individuals. Bait sequences are designed to capture specific alleles previously discovered in parents or reference populations. We studied a set of 135 RILs originating from a cross between an emmer cultivar (Dic2) and a recent durum elite cultivar (Silur). Six thousand sequence baits were designed to target Dic2 vs. Silur polymorphisms discovered in a previous RNAseq study. These baits were exposed to genomic DNA of the RIL population. Eighty percent of the targeted SNPs were recovered, 65% of which were of high quality and coverage. The final high density genetic map consisted of more than 3,000 markers, whose genetic and physical mapping were consistent with those obtained with large arrays.

Familial hypertrophic cardiomyopathy caused by a de novo Gly716Arg mutation of the β-myosin heavy chain.

The present study was performed to identify the genotype of a hypertrophic cardiomyopathy family and investigate the clinicopathogenic characteristics and prognostic features of relevant genetic abnormalities. Target sequence capture sequencing was performed to screen for pathogenic alleles in a 32-year-old female patient (proband). Sanger sequencing was carried out to verify the results. Sanger sequencing was also performed on other family members to identify allele carriers. A survival analysis was carried out using published literature and our findings. We found that the proband and her son harboured a Gly716Arg sequence variant of the β-myosin heavy chain. Neither the proband's father nor the mother were carriers of this sequence variant; thus, the mutation was classified as "de novo". Further survival analysis revealed that female patients appear to have a longer life expectancy compared with males. Our study may provide an effective approach for the genetic diagnosis of hypertrophic cardiomyopathy.