Limits...
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.

Show MeSH
Sequence coverage of target exons enriched in the four chipmunk species. The columns show the distribution of average base coverage per exon. Coverage is shown on the X-axis, binned percentage of exons at each coverage on the Y-axis (left). The green line and right Y-axis show the cumulative coverage as a percent of total exons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472323&req=5

Figure 3: Sequence coverage of target exons enriched in the four chipmunk species. The columns show the distribution of average base coverage per exon. Coverage is shown on the X-axis, binned percentage of exons at each coverage on the Y-axis (left). The green line and right Y-axis show the cumulative coverage as a percent of total exons.

Mentions: The average base coverage within target exons for individual T. alpinus libraries ranged from 7.1 to 13.7X, with a mean of 10.1X ( Additional file 5). 99.6% of the targets had a mean coverage of at least 1X, 89.3% had a mean coverage greater than 5X, and 40.3% had a mean coverage greater than 10X (Figure 3). The average coverage per lane for the other three Tamias species libraries was approximately two times greater than for T. alpinus (25.7X in T. amoenus, 25.9X in T. ruficaudus, and 22.7X in T. striatus), as expected because nearly twice as much sequence data were generated.


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)

Sequence coverage of target exons enriched in the four chipmunk species. The columns show the distribution of average base coverage per exon. Coverage is shown on the X-axis, binned percentage of exons at each coverage on the Y-axis (left). The green line and right Y-axis show the cumulative coverage as a percent of total exons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Sequence coverage of target exons enriched in the four chipmunk species. The columns show the distribution of average base coverage per exon. Coverage is shown on the X-axis, binned percentage of exons at each coverage on the Y-axis (left). The green line and right Y-axis show the cumulative coverage as a percent of total exons.
Mentions: The average base coverage within target exons for individual T. alpinus libraries ranged from 7.1 to 13.7X, with a mean of 10.1X ( Additional file 5). 99.6% of the targets had a mean coverage of at least 1X, 89.3% had a mean coverage greater than 5X, and 40.3% had a mean coverage greater than 10X (Figure 3). The average coverage per lane for the other three Tamias species libraries was approximately two times greater than for T. alpinus (25.7X in T. amoenus, 25.9X in T. ruficaudus, and 22.7X in T. striatus), as expected because nearly twice as much sequence data were generated.

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