Using genic sequence capture in combination with a syntenic pseudo genome to map a deletion mutant in a wheat species

Mapping‐by‐sequencing analyses have largely required a complete reference sequence and employed whole genome re‐sequencing. In species such as wheat, no finished genome reference sequence is available. Additionally, because of its large genome size (17 Gb), re‐sequencing at sufficient depth of coverage is not practical. Here, we extend the utility of mapping by sequencing, developing a bespoke pipeline and algorithm to map an early‐flowering locus in einkorn wheat (Triticum monococcum L.) that is closely related to the bread wheat genome A progenitor. We have developed a genomic enrichment approach using the gene‐rich regions of hexaploid bread wheat to design a 110‐Mbp NimbleGen SeqCap EZ in solution capture probe set, representing the majority of genes in wheat. Here, we use the capture probe set to enrich and sequence an F2 mapping population of the mutant. The mutant locus was identified in T. monococcum, which lacks a complete genome reference sequence, by mapping the enriched data set onto pseudo‐chromosomes derived from the capture probe target sequence, with a long‐range order of genes based on synteny of wheat with Brachypodium distachyon. Using this approach we are able to map the region and identify a set of deleted genes within the interval

A genome-wide survey of DNA methylation in hexaploid wheat

BACKGROUND: DNA methylation is an important mechanism of epigenetic gene expression control that can be passed between generations. Here, we use sodium bisulfite treatment and targeted gene enrichment to study genome-wide methylation across the three sub-genomes of allohexaploid wheat. RESULTS: While the majority of methylation is conserved across all three genomes we demonstrate that differential methylation exists between the sub-genomes in approximately equal proportions. We correlate sub-genome-specific promoter methylation with decreased expression levels and show that altered growing temperature has a small effect on methylation state, identifying a small but functionally relevant set of methylated genes. Finally, we demonstrate long-term methylation maintenance using a comparison between the D sub-genome of hexaploid wheat and its progenitor Aegilops tauschii. CONCLUSIONS: We show that tri-genome methylation is highly conserved with the diploid wheat progenitor while sub-genome-specific methylation shows more variation

Mapping-by-sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat

Previously we extended the utility of mapping-by-sequencing by combining it with sequence capture and mapping sequence data to pseudo-chromosomes that were organized using wheat-Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping-by-synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110MB NimbleGen capture probe set was used to enrich and sequence a doubled-haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo-chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo-chromosomes allow us to demonstrate the application of mapping-by-sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub-genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus - defining a smaller genic region than was previously possible; associate the interval with one wheat sub-genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user-friendly community resource for phenotype mapping

Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome

A modified sequence capture approach allowing standard and methylation analyses of the same enriched genomic DNA sample

Background: Bread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high-quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq. Results: Our method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat. Conclusions: We present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses

Data from: Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this ‘nonmodel’ species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator–activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption

Life without Oxygen: Gene Regulatory Responses of the Crucian Carp (Carassius carassius) Heart Subjected to Chronic Anoxia

Crucian carp are unusual among vertebrates in surviving extended periods in the complete absence of molecular oxygen. During this time cardiac output is maintained though these mechanisms are not well understood. Using a high-density cDNA microarray, we have defined the genome-wide gene expression responses of cardiac tissue after exposing the fish at two temperatures (8 and 13uC) to one and seven days of anoxia, followed by seven days after restoration to normoxia. At 8uC, using a false discovery rate of 5%, neither anoxia nor re-oxygenation elicited appreciable changes in gene expression. By contrast, at 13uC, 777 unique genes responded strongly. Up-regulated genes included those involved in protein turnover, the pentose phosphate pathway and cell morphogenesis while down-regulated gene categories included RNA splicing and transcription. Most genes were affected between one and seven days of anoxia, indicating gene regulation over the medium term but with few early response genes. Re-oxygenation for 7 days was sufficient to completely reverse these responses. Glycolysis displayed more complex responses with anoxia up-regulated transcripts for the key regulatory enzymes, hexokinase and phosphofructokinase, but with down-regulation of most of the non-regulatory genes. This complex pattern of responses in genomic transcription patterns indicates divergent cardiac responses to anoxia, with the transcriptionally driven reprogramming of cardiac function seen at 13uC being largely completed at 8uC

Transcriptomics andin vivotests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this �nonmodel� species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator�activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption

Additional file 1: of A genome-wide survey of DNA methylation in hexaploid wheat

Supplementary data file includes Figures S1–S11, Tables S1–S12 and Notes 1–4. (PDF 107087 kb

Additional file 2: of A modified sequence capture approach allowing standard and methylation analyses of the same enriched genomic DNA sample

Figure S2. Design of the 12 Mbp wheat gene capture array. The 110 Mbp design target sequence for the capture probe set is as described by Gardiner et al. (Gardiner et al., 2015). The RNA baits for this SureSelect Methyl-Seq Target Enrichment system are all 120 bp in length, unique, non-repetitive and are evenly placed across the available wheat genic target sequence according to the design illustrated. (PDF 187 kb