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Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales.

Bi K, Vanderpool D, Singhal S, Linderoth T, Moritz C, Good JM - BMC Genomics (2012)

Bottom Line: There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions.Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

View Article: PubMed Central - HTML - PubMed

Affiliation: Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720-3160, USA. kebi@berkeley.edu

ABSTRACT

Background: To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species.

Results: We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.

Conclusions: Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

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An overall work flow of this study. The Tamias phylogenetic tree is modified from [13] by replacing the outgroup species with T.striatus. The Tamias species that were not under investigation in the present study are not shown.
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Figure 1: An overall work flow of this study. The Tamias phylogenetic tree is modified from [13] by replacing the outgroup species with T.striatus. The Tamias species that were not under investigation in the present study are not shown.

Mentions: In this study, we propose a series of methods (Figure 1) aimed at adapting exon capture based NGS to organisms without pre-existing reference genomes. Here we focused on array-based capture but note that the same general principles should directly extend to an in-solution approach. We focused on North American chipmunks of the genus Tamias to test our methods. Tamias are the focus of a comprehensive set of studies that aim to understand their evolutionary history, patterns of hybridization, and gene introgression (e.g., [13,14]). There is no reference genome currently available for this group; at the onset of our study the most closely related genomic resource was a low-coverage (2X) draft genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), which is around 30 million years (My) divergent from Tamias. The house mouse (Mus musculus) and rat (Rattus norvegicus) are the closest high-quality reference genomes, but last shared a common ancestor with chipmunks around 70 My. In this context, we developed genomic resources by first sequencing multi-tissue transcriptomes from one chipmunk species (the alpine chipmunk, Tamias alpinus), and then designed arrays by targeting a subset of exons from the annotated transcripts. Furthermore, to test how increased divergence affects capture efficiency, we included anonymous genomic targets from the thirteen-lined ground squirrel on this array. We then tested the feasibility of this approach by using these arrays to capture sequence from four chipmunk species, spanning the range of genetic divergence in this genus. Up to 20 individually indexed genomic libraries from each species were pooled and hybridized on single arrays, enabling highly cost-effective sequencing ( Additional file 1) on a scale that is desirable for diverse population genomic and phylogenetic applications. Finally, we developed novel methods for analyzing exon capture data in the absence of a reference genome.


Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales.

Bi K, Vanderpool D, Singhal S, Linderoth T, Moritz C, Good JM - BMC Genomics (2012)

An overall work flow of this study. The Tamias phylogenetic tree is modified from [13] by replacing the outgroup species with T.striatus. The Tamias species that were not under investigation in the present study are not shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472323&req=5

Figure 1: An overall work flow of this study. The Tamias phylogenetic tree is modified from [13] by replacing the outgroup species with T.striatus. The Tamias species that were not under investigation in the present study are not shown.
Mentions: In this study, we propose a series of methods (Figure 1) aimed at adapting exon capture based NGS to organisms without pre-existing reference genomes. Here we focused on array-based capture but note that the same general principles should directly extend to an in-solution approach. We focused on North American chipmunks of the genus Tamias to test our methods. Tamias are the focus of a comprehensive set of studies that aim to understand their evolutionary history, patterns of hybridization, and gene introgression (e.g., [13,14]). There is no reference genome currently available for this group; at the onset of our study the most closely related genomic resource was a low-coverage (2X) draft genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), which is around 30 million years (My) divergent from Tamias. The house mouse (Mus musculus) and rat (Rattus norvegicus) are the closest high-quality reference genomes, but last shared a common ancestor with chipmunks around 70 My. In this context, we developed genomic resources by first sequencing multi-tissue transcriptomes from one chipmunk species (the alpine chipmunk, Tamias alpinus), and then designed arrays by targeting a subset of exons from the annotated transcripts. Furthermore, to test how increased divergence affects capture efficiency, we included anonymous genomic targets from the thirteen-lined ground squirrel on this array. We then tested the feasibility of this approach by using these arrays to capture sequence from four chipmunk species, spanning the range of genetic divergence in this genus. Up to 20 individually indexed genomic libraries from each species were pooled and hybridized on single arrays, enabling highly cost-effective sequencing ( Additional file 1) on a scale that is desirable for diverse population genomic and phylogenetic applications. Finally, we developed novel methods for analyzing exon capture data in the absence of a reference genome.

Bottom Line: There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions.Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

View Article: PubMed Central - HTML - PubMed

Affiliation: Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720-3160, USA. kebi@berkeley.edu

ABSTRACT

Background: To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species.

Results: We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.

Conclusions: Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

Show MeSH