Anchored Hybrid Enrichment data were collected through the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com ) following the general methods of Lemmon et al. [30 (link)] and Prum et al. [59 ]. Briefly, each genomic DNA sample was sonicated to a fragment size of ~175-325 bp using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Subsequently, library preparation and indexing were performed on a Beckman-Coulter Biomek FXp liquid-handling robot following a protocol modified from Meyer and Kircher [60 (link)]. One important modification is a size-selection step after blunt-end repair using SPRIselect beads (Beckman-Coulter Inc.; 0.9x ratio of bead to sample volume). Indexed samples were then pooled at equal quantities (approximately 16 samples per pool), and enrichments were performed on each multi-sample pool using an Agilent Custom SureSelect kit (Agilent Technologies), described herein as the Spider Probe Kit, that contained the probes designed for AHE loci from the spider genomic data detailed above. After enrichment, each set of reactions were pooled in equal quantities for sequencing on three PE150 Illumina HiSeq2500 lanes. Sequencing was performed in the Translational Science Laboratory in the College of Medicine at Florida State University.
Agilent custom sureselect kit
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Custom SureSelect kit is a targeted enrichment solution that allows researchers to design customized panels for sequencing specific genomic regions of interest. The kit provides a streamlined workflow for efficient capture and sequencing of targeted genomic regions, enabling focused analysis and optimization of the regions under investigation.
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5 protocols using agilent custom sureselect kit
1
Spider Probe Kit: Anchored Hybrid Enrichment
2
Multilocus Sequence Data Collection
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Multilocus sequence data were collected at the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com ) following Lemmon et al. [41 (link)] with some adjustments. Each genomic DNA sample was sonicated to a fragment size of ~175–300 bp using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Library preparation and indexing followed Meyer and Kircher [49 (link)]. Indexed libraries were pooled at equal quantities (12 pools of 16 samples each), and the library pools were enriched using a custom Agilent Custom SureSelect kit (Agilent Technologies), with probes designed as described above. The 12 enriched library pools were pooled with equal quantities for sequencing on 4 PE150 Illumina HiSeq2000 lanes with 8 bp indexing. Sequencing was performed at Florida State University in the College of Medicine Translational Science Laboratory.
3
Anchored Phylogenomic Library Preparation
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Data were collected following the general methods of Lemmon et al. [13 (link)] through the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com ). Briefly, 50ul of each genomic DNA sample, with quantity ranging from 11.5 to 985.3 ng) was sonicated to a fragment size of ~150-350 base pairs (bp) using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Subsequently, library preparation and indexing were performed on a Beckman-Coulter Biomek FXp liquid-handling robot following a protocol modified from Meyer and Kirschner [68 (link)]. One important modification is a size-selection step after blunt-end repair using SPRIselect beads (Beckman-Coulter Inc.; 0.9× ratio of bead to sample volume). Indexed samples were then pooled at equal quantities (typically 12–16 samples per pool), and enrichments were performed on each multi-sample pool using an Agilent Custom SureSelect kit (Agilent Technologies), designed as specified above. After enrichment, the three enrichment pools were pooled in equal quantities for sequencing in one PE150 Illumina HiSeq2000 lane. Sequencing was performed in the Translational Science Laboratory in the College of Medicine at Florida State University.
4
Anchored Phylogenomic Sequencing Protocol
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Genetic data were obtained at the Center for Anchored Phylogenomics at Florida State University (http://www.anchoredphylogeny.com ; Last accessed June 22, 2018) using the general methods outlined in Lemmon et al. (2012) (link). First, each genomic DNA sample was sonicated to a fragment size of 150–350 bp using a Covaris E220 focused-ultrasonicator. Subsequently, library preparation and indexing were performed following a protocol outlined in (Meyer and Kircher 2010 (link)). Indexed samples were then pooled at equal quantities determined using a Qubit fluorometer (16 samples per pool), and samples in each pool were enriched using an Agilent Custom SureSelect kit (Agilent Technologies) with custom designed probes. The general enrichment process uses streptavidin-coated magnetic beads to isolate targeted genomic fragments that hybridize with the oligonucleotide probes (Gnirke et al. 2009 (link)). Enriched fragments were pooled in groups of three (three pools total with 48 samples in each) before sequencing on the PE150 Illumina HiSeq2500 (three lanes, 48 samples per lane). Sequencing was performed in the Translational Science Laboratory in the College of Medicine at Florida State University.
5
Diptera Targeted Enrichment Protocol
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For each sample, 7.9–110 ng/μL (47 ng/μL mean) DNA in 50 μL total volume was sheared to approximately 300 bp by sonication with a Covaris E220 Focused-ultrasonicator using Covaris microTUBES (Covaris, Inc., MA, USA). The sheared DNA was used as input for genomic DNA library preparation and indexing using the protocol of Meyer & Kircher [80 (link)], but modified to include a size-selection step after blunt-end repair using SPRIselect beads (Beckman Coulter, Inc., CA, USA; 0.9 × ratio of bead to sample volume). Each sample was then indexed and pooled together in groups of 48 samples. We enriched each 48-sample pool using the 57, 681 tiled, custom-designed probes contained in the Diptera AHE kit [75 (link)], an Agilent Custom SureSelect kit (Agilent Technologies, CA, USA) that targets 559 unique loci. The Diptera probe kit design is detailed in Young et al. [75 (link)] and is based on comparison and selection of conserved 150 bp gene regions found among seven diverse fly genomes and 14 transcriptomes. We sequenced the pooled libraries using two lanes of an Illumina HiSeq 2500 (Illumina, CA, USA) run (single read, 100 bp). All AHE laboratory procedures and sequencing were conducted in laboratory facilities of the North Carolina State University (NCSU), Department of Entomology and Plant Pathology (Wiegmann Lab) and the NCSU Genomic Sciences Laboratory (GSL).
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