Limits...
Targeted Capture of Phylogenetically Informative Ves SINE Insertions in Genus Myotis.

Platt RN, Zhang Y, Witherspoon DJ, Xing J, Suh A, Keith MS, Jorde LB, Stevens RD, Ray DA - Genome Biol Evol (2015)

Bottom Line: To address this problem, we have extended a retrotransposon-based capture and sequence method (ME-Scan [mobile element scanning]) to identify insertions belonging to the Ves family of short interspersed elements (SINEs) across seven species of the bat genus Myotis.This phylogeny is similar to previously published mitochondrial phylogenies, with the exception of the placement of M. vivesi.These results support the utility of our variation on ME-Scan to identify polymorphic retrotransposon insertions in taxa without a reference genome and for large-scale retrotransposon-based phylogenetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University Department of Biological Sciences, Texas Tech University.

Show MeSH
Preparation and sequencing of Ves enriched libraries. (A) gDNA is fragmented to an average size of 1 kb. (B) The fragmented, gDNA is end-repaired, and dA tailed before indexed adapters are added. (C) Individual libraries are pooled together and DNA fragments containing Ves are bound with a biotinylated Ves probe using a five-cycle PCR reaction. (D) The entire library is size-sorted along a gel. Fragments 500–600 bp in length are isolated. (E) The size-selected fragments are enriched for Ves by binding the Ves biotinylated probe to streptavidin-coated magnetic beads. (F) A final amplification of 15–20 cycles is used to amplify the library of Ves-enriched fragments for sequencing. (G) A final library of 550-bp Ves fragments is isolated through gel electrophoresis and purified for sequencing. (H) Fragments are sequenced so that one read contains the species-specific index plus DNA sequence up to 300–400 bp away from the Ves insertion (flanking read). The second read contains a portion of the Ves insertion plus approximately 80 bp of the Ves insertion site (Ves read).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494050&req=5

evv099-F1: Preparation and sequencing of Ves enriched libraries. (A) gDNA is fragmented to an average size of 1 kb. (B) The fragmented, gDNA is end-repaired, and dA tailed before indexed adapters are added. (C) Individual libraries are pooled together and DNA fragments containing Ves are bound with a biotinylated Ves probe using a five-cycle PCR reaction. (D) The entire library is size-sorted along a gel. Fragments 500–600 bp in length are isolated. (E) The size-selected fragments are enriched for Ves by binding the Ves biotinylated probe to streptavidin-coated magnetic beads. (F) A final amplification of 15–20 cycles is used to amplify the library of Ves-enriched fragments for sequencing. (G) A final library of 550-bp Ves fragments is isolated through gel electrophoresis and purified for sequencing. (H) Fragments are sequenced so that one read contains the species-specific index plus DNA sequence up to 300–400 bp away from the Ves insertion (flanking read). The second read contains a portion of the Ves insertion plus approximately 80 bp of the Ves insertion site (Ves read).

Mentions: A summary of the biochemical pipeline is shown in figure 1A–H. Genomic DNA (gDNA) was extracted from tissue samples using a standard phenol–chloroform/ethanol precipitation protocol. For each sample, 10 µg of gDNA was fragmented to an average size of 1 kb on a Covaris S220 Focused-ultrasonicator using the following parameters: Peak incident power, 105 W; duty factor, 5.0%; cycles per burst, 200; time, 40 s; temperature, 7 °C (fig. 1A). Fragmented gDNA was purified and concentrated using Qiagen QIAquick PCR purification columns (Qiagen, Germantown, MD) using the recommended protocol.Fig. 1.—


Targeted Capture of Phylogenetically Informative Ves SINE Insertions in Genus Myotis.

Platt RN, Zhang Y, Witherspoon DJ, Xing J, Suh A, Keith MS, Jorde LB, Stevens RD, Ray DA - Genome Biol Evol (2015)

Preparation and sequencing of Ves enriched libraries. (A) gDNA is fragmented to an average size of 1 kb. (B) The fragmented, gDNA is end-repaired, and dA tailed before indexed adapters are added. (C) Individual libraries are pooled together and DNA fragments containing Ves are bound with a biotinylated Ves probe using a five-cycle PCR reaction. (D) The entire library is size-sorted along a gel. Fragments 500–600 bp in length are isolated. (E) The size-selected fragments are enriched for Ves by binding the Ves biotinylated probe to streptavidin-coated magnetic beads. (F) A final amplification of 15–20 cycles is used to amplify the library of Ves-enriched fragments for sequencing. (G) A final library of 550-bp Ves fragments is isolated through gel electrophoresis and purified for sequencing. (H) Fragments are sequenced so that one read contains the species-specific index plus DNA sequence up to 300–400 bp away from the Ves insertion (flanking read). The second read contains a portion of the Ves insertion plus approximately 80 bp of the Ves insertion site (Ves read).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494050&req=5

evv099-F1: Preparation and sequencing of Ves enriched libraries. (A) gDNA is fragmented to an average size of 1 kb. (B) The fragmented, gDNA is end-repaired, and dA tailed before indexed adapters are added. (C) Individual libraries are pooled together and DNA fragments containing Ves are bound with a biotinylated Ves probe using a five-cycle PCR reaction. (D) The entire library is size-sorted along a gel. Fragments 500–600 bp in length are isolated. (E) The size-selected fragments are enriched for Ves by binding the Ves biotinylated probe to streptavidin-coated magnetic beads. (F) A final amplification of 15–20 cycles is used to amplify the library of Ves-enriched fragments for sequencing. (G) A final library of 550-bp Ves fragments is isolated through gel electrophoresis and purified for sequencing. (H) Fragments are sequenced so that one read contains the species-specific index plus DNA sequence up to 300–400 bp away from the Ves insertion (flanking read). The second read contains a portion of the Ves insertion plus approximately 80 bp of the Ves insertion site (Ves read).
Mentions: A summary of the biochemical pipeline is shown in figure 1A–H. Genomic DNA (gDNA) was extracted from tissue samples using a standard phenol–chloroform/ethanol precipitation protocol. For each sample, 10 µg of gDNA was fragmented to an average size of 1 kb on a Covaris S220 Focused-ultrasonicator using the following parameters: Peak incident power, 105 W; duty factor, 5.0%; cycles per burst, 200; time, 40 s; temperature, 7 °C (fig. 1A). Fragmented gDNA was purified and concentrated using Qiagen QIAquick PCR purification columns (Qiagen, Germantown, MD) using the recommended protocol.Fig. 1.—

Bottom Line: To address this problem, we have extended a retrotransposon-based capture and sequence method (ME-Scan [mobile element scanning]) to identify insertions belonging to the Ves family of short interspersed elements (SINEs) across seven species of the bat genus Myotis.This phylogeny is similar to previously published mitochondrial phylogenies, with the exception of the placement of M. vivesi.These results support the utility of our variation on ME-Scan to identify polymorphic retrotransposon insertions in taxa without a reference genome and for large-scale retrotransposon-based phylogenetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University Department of Biological Sciences, Texas Tech University.

Show MeSH