|
| Status |
Public on Apr 07, 2014 |
| Title |
Sexual_Plant8-Cy5 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Sexual_Plant8_CGH
|
| Organism |
Boechera stricta |
| Characteristics |
ipk-plant id: B10-851
name: Sex-8
es numbera: ES 661
species: B. stricta
reproduction: Sexual
locality: Golden Gate B
u.s. state: CO
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was extracted from the leaf tissue of a single plant of each of the 20 genotypes using a modified CTAB method described by Mace et al. (2003). Plant Mol. Biol. Report 21:459a-459h.
|
| Label |
Cy5
|
| Label protocol |
DNA with concentrations between 450 ng/μl and 1.0 μg/μl and absorbance readings of A260/A280 > 1.8 and A260/A230 > 1.9 (recommended by Roche-NimbleGen) were used for the labeling procedure. A total of 1.0 µg of gDNA per sample was used for each labeling reaction and hybridization thereafter. A dye swap experimental design was used for technical replication, where both test and reference samples are interchangeably labeled with the two dyes (Cy3 and Cy5; following Roche-NimblGen standard protocols) in order to minimize error due to dye-bias. A pooled reference sample was used, whereby an equal concentration of gDNA from each of the 20 genotypes was combined as a reference in each labeling reaction and hybridization (Scherer et al. 2007, Nat. Genet. 39:S7–S15.; Ju et al. 2010, Nucleic Acids Res. 1–11, doi:10.1093/nar/gkq730).
|
| |
|
| Channel 2 |
| Source name |
pooled reference sample (mixture of different genotypes)
|
| Organisms |
Boechera stricta; Boechera lignifera; Boechera divaricarpa; Boechera crandallii; Boechera retrofracta; Boechera schistacea; Boechera polyantha; Boechera retrofracta x Boechera stricta; Boechera duchesnensis |
| Characteristics |
reference: mixture of all 20 genotypes
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was extracted from the leaf tissue of a single plant of each of the 20 genotypes using a modified CTAB method described by Mace et al. (2003). Plant Mol. Biol. Report 21:459a-459h.
|
| Label |
Cy3
|
| Label protocol |
DNA with concentrations between 450 ng/μl and 1.0 μg/μl and absorbance readings of A260/A280 > 1.8 and A260/A230 > 1.9 (recommended by Roche-NimbleGen) were used for the labeling procedure. A total of 1.0 µg of gDNA per sample was used for each labeling reaction and hybridization thereafter. A dye swap experimental design was used for technical replication, where both test and reference samples are interchangeably labeled with the two dyes (Cy3 and Cy5; following Roche-NimblGen standard protocols) in order to minimize error due to dye-bias. A pooled reference sample was used, whereby an equal concentration of gDNA from each of the 20 genotypes was combined as a reference in each labeling reaction and hybridization (Scherer et al. 2007, Nat. Genet. 39:S7–S15.; Ju et al. 2010, Nucleic Acids Res. 1–11, doi:10.1093/nar/gkq730).
|
| |
|
| |
| Hybridization protocol |
A total of 1.0 µg of gDNA per sample was used for each labeling reaction and hybridization thereafter. A dye swap experimental design was used for technical replication, where both test and reference samples are interchangeably labeled with the two dyes (Cy3 and Cy5; following Roche-NimblGen standard protocols) in order to minimize error due to dye-bias. A pooled reference sample was used, whereby an equal concentration of gDNA from each of the 20 genotypes was combined as a reference in each labeling reaction and hybridization (Scherer et al. 2007, Nat. Genet. 39:S7–S15.; Ju et al. 2010, Nucleic Acids Res. 1–11, doi:10.1093/nar/gkq730).
|
| Scan protocol |
After hybridization, washing and drying (following the manufacturer’s protocols; Roche-NimblGen) the arrays were scanned at 2 µm double pass resolution with an Agilent Microarray G2565BA Fluorescent Scanner (Agilent Technologies). Each 3-plex image (3x720K) was split into three separate arrays and aligned for data extraction using the NimbleScan software (version 2.6; Roche-NimblGen). The NimbleScan software performs spatial correction of the data, thereby reducing artifacts and noise before applying q-spline fit normalization (Workman et al. 2002). Data in *unavg_segMNT.gff files represent raw data as coming from the NimbleScan software.
|
| Data processing |
For the dye-swap experimental design, two CGH profiles (Cy3 and Cy5) were obtained, each representing the log2-ratios of measured fluorescence intensities for the test (sexual or apomictic genotype; Table 1) versus reference sample (mixture of different genotypes, see above) for all 719 346 probes on the array (720K minus control probes). Genome-wide CGH-profiles of each genotype were normalized by applying quantile normalization (Bolstad et al. 2003) to both CGH profiles from the dye swap.
All normalized CGH profiles were analyzed by a mixture model of three Gaussian distributions described in Seifert et al. (2012, PLoS Comp. Biol. 8: doi: 10.1371/journal.pcbi.1002286 ) to identify depletions, enrichment and unchanged regions between a test genotype and the reference sample by specifically adapting the parameters of the mixture model to both CGH profiles of each test genotype using a Bayesian Expectation Maximization (EM) algorithm. Identification of depleted, unchanged, and enriched regions was done by performing a decoding of the measured log2-ratios into the most likely underlying state of each probe (depleted, unchanged, or enriched). Only probes that were identified as depleted or enriched in both CGH profiles (dye swap replicates) of a test sample (genotype) were considered as being depleted or enriched in this genotype, while all other probes were considered as being unchanged (i.e. no CNV variation with respect to the pooled reference). This resulted in a reproducible set of genomic regions affected by depletions or enrichment for each genotype.
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| |
|
| Submission date |
Apr 04, 2014 |
| Last update date |
Apr 07, 2014 |
| Contact name |
Timothy Sharbel |
| E-mail(s) |
sharbel@ipk-gatersleben.de
|
| Phone |
+49394825608
|
| Organization name |
IPK Gatersleben
|
| Street address |
Corrensstrasse 4
|
| City |
Gatersleben |
| ZIP/Postal code |
06466 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17827 |
| Series (1) |
| GSE51596 |
CNV in sexual and apomictic Boechera |
|
