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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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Coverage-exon distance distributions. Exons that ranged between 201–600 bp were used for generating the plot. Each target exon was split into 20-bp bins depicted by the red bar (X-axis). The average base coverage within each exon bin is shown on the Y-axis.
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Figure 4: Coverage-exon distance distributions. Exons that ranged between 201–600 bp were used for generating the plot. Each target exon was split into 20-bp bins depicted by the red bar (X-axis). The average base coverage within each exon bin is shown on the Y-axis.

Mentions: For each target exon, we found that the coverage among bases was mostly uniform except for at the edges. For example, base coverage for T. alpinus exons increased asymptotically from 5.5X at the 5’ and 3’ ends of exons towards the center were it reached ~12X and plateaued at 80–100 bp from the ends (Figure 4). A major limitation of transcriptome-based array design is the absence of tiling probes that span exon-intron boundaries, and fewer probes tiled at the ends of exons results in reduced coverage of contig edges. To accommodate this shortfall, we applied extra tiling probes to the edges of each exon to promote more uniform coverage. However, our results indicate that this method did not completely solve the expected “edge effect”. To address the edge effect completely, future array designs could use denser tiling even further from the edge and/or print duplicate probes to target the first or last few bases of each exon.


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)

Coverage-exon distance distributions. Exons that ranged between 201–600 bp were used for generating the plot. Each target exon was split into 20-bp bins depicted by the red bar (X-axis). The average base coverage within each exon bin is shown on the Y-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Coverage-exon distance distributions. Exons that ranged between 201–600 bp were used for generating the plot. Each target exon was split into 20-bp bins depicted by the red bar (X-axis). The average base coverage within each exon bin is shown on the Y-axis.
Mentions: For each target exon, we found that the coverage among bases was mostly uniform except for at the edges. For example, base coverage for T. alpinus exons increased asymptotically from 5.5X at the 5’ and 3’ ends of exons towards the center were it reached ~12X and plateaued at 80–100 bp from the ends (Figure 4). A major limitation of transcriptome-based array design is the absence of tiling probes that span exon-intron boundaries, and fewer probes tiled at the ends of exons results in reduced coverage of contig edges. To accommodate this shortfall, we applied extra tiling probes to the edges of each exon to promote more uniform coverage. However, our results indicate that this method did not completely solve the expected “edge effect”. To address the edge effect completely, future array designs could use denser tiling even further from the edge and/or print duplicate probes to target the first or last few bases of each exon.

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