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Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • METHODS AND RESULTS:Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • CONCLUSIONS:The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

SUBMITTER: Weitemier K

PROVIDER: S-EPMC4162667 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

Weitemier Kevin K Straub Shannon C K SC Cronn Richard C RC Fishbein Mark M Schmickl Roswitha R McDonnell Angela A Liston Aaron A

Applications in plant sciences 20140829 9

<h4>Unlabelled</h4><h4>Premise of the study</h4>Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. •<h4>Methods and results</h4>Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 i ...[more]

PMID: 25225629

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Project description:Targeted enrichment and sequencing of hundreds of nuclear loci for phylogenetic reconstruction is becoming an important tool for plant systematics and evolution. Annonaceae is a major pantropical plant family with 110 genera and ca. 2,450 species, occurring across all major and minor tropical forests of the world. Baits were designed by sequencing the transcriptomes of five species from two of the largest Annonaceae subfamilies. Orthologous loci were identified. The resulting baiting kit was used to reconstruct phylogenetic relationships at two different levels using concatenated and gene tree approaches: a family wide Annonaceae analysis sampling 65 genera and a species level analysis of tribe Piptostigmateae sampling 29 species with multiple individuals per species. DNA extraction was undertaken mainly on silicagel dried leaves, with two samples from herbarium dried leaves. Our kit targets 469 exons (364,653 bp of sequence data), successfully capturing sequences from across Annonaceae. Silicagel dried and herbarium DNA worked equally well. We present for the first time a nuclear gene-based phylogenetic tree at the generic level based on 317 supercontigs. Results mainly confirm previous chloroplast based studies. However, several new relationships are found and discussed. We show significant differences in branch lengths between the two large subfamilies Annonoideae and Malmeoideae. A new tribe, Annickieae, is erected containing a single African genus Annickia. We also reconstructed a well-resolved species-level phylogenetic tree of the Piptostigmteae tribe. Our baiting kit is useful for reconstructing well-supported phylogenetic relationships within Annonaceae at different taxonomic levels. The nuclear genome is mainly concordant with plastome information with a few exceptions. Moreover, we find that substitution rate heterogeneity between the two subfamilies is also found within the nuclear compartment, and not just plastomes and ribosomal DNA as previously shown. Our results have implications for understanding the biogeography, molecular dating and evolution of Annonaceae.

Project description:DNA CpG methylation is a widespread epigenetic mark in high eukaryotes including mammals. DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and control of gene expression. Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to measure DNA methylation in a genome-wide fashion, making it possible to comprehensively investigate the role of DNA methylation. Several methods have been developed, such as Whole Genome Bisulfite Sequencing (WGBS), Reduced Representation Bisulfite Sequencing (RRBS), and enrichment-based methods including Methylation Dependent ImmunoPrecipitation followed by sequencing (MeDIP-seq), methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), methyltransferase-directed Transfer of Activated Groups followed by sequencing (mTAG), and Methylation-sensitive Restriction Enzyme digestion followed by sequencing (MRE-seq). These methods differ by their genomic CpG coverage, resolution, quantitative accuracy, cost, and software for analyzing the data. Among these, WGBS is considered the gold standard. However, it is still a cost-prohibitive technology for a typical laboratory due to the required sequencing depth. We found that by integrating two enrichment-based methods that are complementary in nature (i.e., MeDIP-seq and MRE-seq), we can significantly increase the efficiency of whole DNA methylome profiling. By using two recently developed computational algorithms (i.e., M&M and methylCRF), the combination of MeDIP-seq and MRE-seq produces genome-wide CpG methylation measurement at high coverage and high resolution, and robust predictions of differentially methylated regions. Thus, the combination of the two enrichment-based methods provides a cost-effective alternative to WGBS. In this article we describe both the experimental protocols for performing MeDIP-seq and MRE-seq, and the computational protocols for running M&M and methylCRF.

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Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

Publications

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

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