Exon markers have a long history of use in phylogenetics of ray‐finned fishes, the most diverse clade of vertebrates with more than 35,000 species. As the number of published genomes increases, it has become easier to test exons and other genetic markers for signals of ancient duplication events and filter out paralogues that can mislead phylogenetic analysis. We present seven new probe sets for current target‐capture phylogenomic protocols that capture 1,104 exons explicitly filtered for paralogues using gene trees. These seven probe sets span the diversity of teleost fishes, including four sets that target five hyperdiverse percomorph clades which together comprise ca. 17,000 species (Carangaria, Ovalentaria, Eupercaria, and Syngnatharia + Pelagiaria combined). We additionally included probes to capture legacy nuclear exons and mitochondrial markers that have been commonly used in fish phylogenetics (despite some exons being flagged for paralogues) to facilitate integration of old and new molecular phylogenetic matrices. We tested these probes experimentally for 56 fish species (eight species per probe set) and merged new exon‐capture sequence data into an existing data matrix of 1,104 exons and 300 ray‐finned fish species. We provide an optimized bioinformatics pipeline to assemble exon capture data from raw reads to alignments for downstream analysis. We show that legacy loci with known paralogues are at risk of assembling duplicated sequences with target‐capture, but we also assembled many useful orthologous sequences that can be integrated with many PCR‐generated matrices. These probe sets are a valuable resource for advancing fish phylogenomics because targeted exons can easily be extracted from increasingly available whole genome and transcriptome data sets, and also may be integrated with existing PCR‐based exon and mitochondrial data.
Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome‐based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron–exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome‐based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in
- PAR ID:
- 10243953
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 16
- Issue:
- 5
- ISSN:
- 1755-098X
- Page Range / eLocation ID:
- p. 1069-1083
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Exons within transcripts are traditionally classified as first, internal or last exons, each governed by different regulatory mechanisms. We recently described the widespread usage of ‘hybrid’ exons that serve as terminal or internal exons in different transcripts. Here, we employ an interpretable deep learning pipeline to dissect the sequence features governing the co-regulation of transcription initiation and splicing in hybrid exons. Using ENCODE data from human tissues, we identified 80 000 hybrid first-internal exons. These exons often possess a relaxed chromatin state, allowing transcription initiation within the gene body. Interestingly, transcription start sites of hybrid exons are typically centered at the 3′ splice site, suggesting tight coupling between splicing and transcription initiation. We identified two subcategories of hybrid exons: the majority resemble internal exons, maintaining strong 3′ splice sites, while a minority show enrichment in promoter elements, resembling first exons. Diving into the evolution of their sequences, we found that human hybrid exons with orthologous first exons in other species usually gained 3′ splice sites or whole exons upstream, while those with orthologous internal exons often gained promoter elements. Overall, our findings unveil the intricate regulatory landscape of hybrid exons and reveal stronger connections between transcription initiation and RNA splicing than previously acknowledged.
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Abstract Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating
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