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Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements.

ABSTRACT: Advances in high-throughput sequencing techniques now allow relatively easy and affordable sequencing of large portions of the genome, even for nonmodel organisms. Many phylogenetic studies reduce costs by focusing their sequencing efforts on a selected set of targeted loci, commonly enriched using sequence capture. The advantage of this approach is that it recovers a consistent set of loci, each with high sequencing depth, which leads to more confidence in the assembly of target sequences. High sequencing depth can also be used to identify phylogenetically informative allelic variation within sequenced individuals, but allele sequences are infrequently assembled in phylogenetic studies. Instead, many scientists perform their phylogenetic analyses using contig sequences which result from the de novo assembly of sequencing reads into contigs containing only canonical nucleobases, and this may reduce both statistical power and phylogenetic accuracy. Here, we develop an easy-to-use pipeline to recover allele sequences from sequence capture data, and we use simulated and empirical data to demonstrate the utility of integrating these allele sequences to analyses performed under the multispecies coalescent model. Our empirical analyses of ultraconserved element locus data collected from the South American hummingbird genus Topaza demonstrate that phased allele sequences carry sufficient phylogenetic information to infer the genetic structure, lineage divergence, and biogeographic history of a genus that diversified during the last 3 myr. The phylogenetic results support the recognition of two species and suggest a high rate of gene flow across large distances of rainforest habitats but rare admixture across the Amazon River. Our simulations provide evidence that analyzing allele sequences leads to more accurate estimates of tree topology and divergence times than the more common approach of using contig sequences.

SUBMITTER: Andermann T

PROVIDER: S-EPMC6292485 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements.

Andermann Tobias T Fernandes Alexandre M AM Olsson Urban U Töpel Mats M Pfeil Bernard B Oxelman Bengt B Aleixo Alexandre A Faircloth Brant C BC Antonelli Alexandre A

Systematic biology 20190101 1

Advances in high-throughput sequencing techniques now allow relatively easy and affordable sequencing of large portions of the genome, even for nonmodel organisms. Many phylogenetic studies reduce costs by focusing their sequencing efforts on a selected set of targeted loci, commonly enriched using sequence capture. The advantage of this approach is that it recovers a consistent set of loci, each with high sequencing depth, which leads to more confidence in the assembly of target sequences. High ...[more]

PMID: 29771371

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Project description:Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2-121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6-972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06-9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21-39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens.

Project description:BackgroundOligodendrocytes are specialized cells of the nervous system that produce the myelin sheaths surrounding the axons of neurons. Myelinating the axons increases the speed of nerve conduction and demyelination contributes to the pathology of neurodegenerative diseases such as multiple sclerosis. Oligodendrocyte differentiation is specified early in development by the expression of the basic-helix-loop-helix transcription factor Olig2 in the ventral region of the neural tube. Understanding how Olig2 expression is controlled is therefore essential for elucidating the mechanisms governing oligodendrocyte differentiation. A method is needed to identify potential regulatory sequences in the long stretches of adjacent non-coding DNA that flank Olig2.Methodology/principal findingsWe identified ten potential regulatory regions upstream of Olig2 based on a combination of bioinformatics metrics that included evolutionary conservation across multiple vertebrate genomes, the presence of potential transcription factor binding sites and the existence of ultraconserved elements. One of our computational predictions includes a region previously identified as the Olig2 basal promoter, suggesting that our criterion represented characteristics of known regulatory regions. In this study, we tested one candidate regulatory region for its ability to modulate the Olig2 basal promoter and found that it represses expression in undifferentiated embryonic stem cells.Conclusions/significanceThe regulatory region we identified modifies the expression regulated by the Olig2 basal promoter in a manner consistent with our current understanding of Olig2 expression during oligodendrocyte differentiation. Our results support a model in which constitutive activation of Olig2 by its basal promoter is repressed in undifferentiated cells by upstream repressive elements until that repression is relieved during differentiation. We conclude that the potential regulatory elements presented in this study provide a good starting point for unraveling the cis-regulatory logic that governs Olig2 expression. Future studies of the functionality of the potential regulatory elements we present will help reveal the interactions that govern Olig2 expression during development.

Dataset Information

Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements.

Publications

Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements.

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