Dataset Information

Development of novel, exon-primed intron-crossing (EPIC) markers from EST databases and evaluation of their phylogenetic utility in Commiphora (Burseraceae).

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Novel nuclear exon-primed intron-crossing (EPIC) markers were developed to increase phylogenetic resolution among recently diverged lineages in the frankincense and myrrh family, Burseraceae, using Citrus, Arabidopsis, and Oryza genome resources. • METHODS AND RESULTS:Primer pairs for 48 nuclear introns were developed using the genome resource IntrEST and were screened using species of Commiphora and other Burseraceae taxa. Four putative intron regions (RPT6A, BXL2, mtATP Synthase D, and Rab6) sequenced successfully for multiple taxa and recovered phylogenies consistent with those of existing studies. In some cases, these regions yielded informative sequence variation on par with that of the nuclear ribosomal DNA internal transcribed spacer. • CONCLUSIONS:The combination of freely available genome resources and our design criteria have uncovered four single-copy nuclear intron regions that are useful for phylogenetic reconstruction of Burseraceae taxa. Because our EPIC primers also amplify Arabidopsis, we recommend their trial in other rosid and eudicot lineages.

SUBMITTER: Gostel MR

PROVIDER: S-EPMC4103138 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

Development of novel, exon-primed intron-crossing (EPIC) markers from EST databases and evaluation of their phylogenetic utility in Commiphora (Burseraceae).

Gostel Morgan R MR Weeks Andrea A

Applications in plant sciences 20140331 4

<h4>Unlabelled</h4><h4>Premise of the study</h4>Novel nuclear exon-primed intron-crossing (EPIC) markers were developed to increase phylogenetic resolution among recently diverged lineages in the frankincense and myrrh family, Burseraceae, using Citrus, Arabidopsis, and Oryza genome resources. •<h4>Methods and results</h4>Primer pairs for 48 nuclear introns were developed using the genome resource IntrEST and were screened using species of Commiphora and other Burseraceae taxa. Four putative int ...[more]

PMID: 25202619

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Project description:BACKGROUND: Exon-primed intron-crossing (EPIC) markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene sequences, particularly in teleost fishes. RESULTS: We introduce a bioinformatics pipeline for developing EPIC markers by comparing the whole genome sequences between two or more species. By applying this approach on five teleost fishes whose genomes were available in the Ensembl database http://www.ensembl.org, we identified 210 EPIC markers that have single-copy and conserved exon regions with identity greater than 85% among the five teleost fishes. We tested 12 randomly chosen EPIC markers in nine teleost species having a wide phylogenetic range. The success rate of amplifying and sequencing those markers varied from 44% to 100% in different species. We analyzed the exon sequences of the 12 EPIC markers from 13 teleosts. The resulting phylogeny contains many traditionally well-supported clades, indicating the usefulness of the exon portion of EPIC markers in reconstructing species phylogeny, in addition to the value of the intron portion of EPIC markers in interrogating the population history. CONCLUSIONS: This study illustrated an effective approach to develop EPIC markers in a taxonomic group, where two or more genome sequences are available. The markers identified could be amplified across a broad taxonomic range of teleost fishes. The phylogenetic utility of individual markers varied according to intron size and amplifiability. The bioinformatics pipelines developed are readily adapted to other taxonomic groups.

Project description:BACKGROUND:In-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases. RESULTS:We present IntEREst, Intron-Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies. CONCLUSION:IntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns.

Dataset Information

Development of novel, exon-primed intron-crossing (EPIC) markers from EST databases and evaluation of their phylogenetic utility in Commiphora (Burseraceae).

Publications

Development of novel, exon-primed intron-crossing (EPIC) markers from EST databases and evaluation of their phylogenetic utility in Commiphora (Burseraceae).

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