|
| Status |
Public on Nov 25, 2009 |
| Title |
T3BQBC06182008A_3156_CY3_STERNE_CY5_13824082_B |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
2008723156
|
| Organism |
Bacillus cereus |
| Characteristics |
strain: 2008723156
|
| Biomaterial provider |
Dr. A. Hoffmaster, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
|
| Treatment protocol |
None.
|
| Growth protocol |
Cells grown in Lysogeny broth (LB) at 37 degrees Celcius overnight.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Epicentre MasterPure DNA Extraction Kit.
|
| Label |
Cy3
|
| Label protocol |
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with the Qiaquick PCR purification kit (QIAGEN). The purified labelled DNA was then dye coupled with the appropriate cyanine dye (Cy3/Cy5).
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| |
|
| Channel 2 |
| Source name |
Sterne reference
|
| Organism |
Bacillus anthracis str. Sterne |
| Characteristics |
strain: Sterne
|
| Biomaterial provider |
Dr. A. Hoffmaster, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
|
| Treatment protocol |
None.
|
| Growth protocol |
Cells grown in Lysogeny broth (LB) at 37 degrees Celcius overnight.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Epicentre MasterPure DNA Extraction Kit.
|
| Label |
Cy5
|
| Label protocol |
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with the Qiaquick PCR purification kit (QIAGEN). The purified labelled DNA was then dye coupled with the appropriate cyanine dye (Cy3/Cy5).
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| |
|
| |
| Hybridization protocol |
Aminosaline coated slides were prehybridized in 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (Invitrogen), 0.1% sodium dodecyl sulfate, and 1% bovine serum albumin at 42 degree Celcius for 45 min. The slides then were washed at room temperature with distilled water, dipped in isopropanol, and spun dry. Equal concentrations of DNA probe from the appropriate Cy3 and Cy5 labeled probes were combined, dried and then resuspended in a solution of 35% formamide, 5x SSC, and 0.1% sodium dodecyl sulfate. Resuspended probes were denatured at 95 degree Celcius prior to hybridization. The probe mixture then was added to the microarray slide and allowed to hybridize overnight at 42 degree Celcius. Hybridized slides were washed sequentially in solutions of 1x SSC-0.2% SDS, 0.1x SSC-0.2% SDS, and 0.1x SSC at room temperature.
|
| Scan protocol |
Scanned on Axon GenePix 4000 scanner. PMT values were optimized during scanning to balance channel intensities.
|
| Description |
T3BQBC06182008A_3156_CY3_STERNE_CY5_13824082_anno_sub_95th_MDS.mev.refIsMEDB.out
|
| Data processing |
Procedures to calculate the value of log2 ratio:
1. Use software suite TM4 (www.tm4.org) to analyze spotted arrays.
2. Use Spotfinder with background correction to compute the spot intensities of the hybridized arrays.
3. Perform the following filtering steps:
All PFGRC arrays used in this study have 3 types of spots.
(1). Species spot: oligos designed for the species under investigation.
(2). Reserved spot or control spot: oligos designed from Arabidopsis genes as controls.
(3). Empty spots: no oligos or any DNA.
Step 1: Identify the replicates.
Sort the .mev file from Spotfinder by oligo_id.
Step 2: Identify the outliers within each set of replicates for each channel.
Outliers are identified using the Sprents method (The Handbook of Parametric and Nonparametric Statistical Procedures, Third Edition by David J. Sheskin, PP 400-402).
|Xi - M|/MAD > Max
Xi = Each intensity value from the set of replicates.
M = Median intensity value from the set of replicates.
MAD = Median absolute deviation.
Max = the critical value that the result of the inequality must exceed in order to conclude that the Xi value is an outlier. The value of 5 was used for Max.
Step 3: Replace the outliers.
Replace the outliers by the value closest to them from the tail that they occur. For example, if the outlier identified is the highest value in channel A, that value will be replaced by the next highest value of its replicate set in channel A.
Step 4: Subtract the 95th percentile of the reserved spots.
Calculate the 95th percentile of all the reserved spots and subtract that value from all the species spots and reserved spots.
Step 5: Replace zero (0) by one (1).
After subtracting the 95th percentile of the reserved spots, if one of the channel intensities is less than zero (0), replace that value by one (1); If both channels become less than zero (0), set both channels to zero (0).
Step 6. Output the filtered .mev file.
4. Use Midas X for CGH normalization:
1). Plot the histogram of the Log2 ratio data (Reference/Query) from the filtered .mev file, and identify the central peak containing oligos predicted to be shared by both the reference and query strains.
2). Fit a Gaussian curve to the central peak by 'tweaking' the log ratio mean and by shrinking or stretching the standard deviation of the curve to find the smallest least sum of squares difference.
3). Perform Log Mode Centering normalization on the channel that contains the query sample only (Either Cy3 or Cy5).
4). Output the normalized *.mev file.
5. Calculate the value of log2 ratio (Reference/Query) from the normalized .mev file:
1). If both channels (QUERY_MEDIAN_INTENSITY and REF_MEDIAN_INTENSITY) are zero, assign the value "null".
2). If one channel is zero and the other is not zero, substitute the zero with one (1) and then, assign the log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY to VALUE
3). If neither channel is zero, assign the log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY to VALUE.
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| |
|
| Submission date |
Nov 13, 2009 |
| Last update date |
Nov 24, 2009 |
| Contact name |
Chun-Hua Wan |
| E-mail(s) |
cwan@jcvi.org
|
| Phone |
301-795-7707
|
| Organization name |
The J. Craig Venter Institute
|
| Department |
Pathogen Functional Genomics Resources Center (PFGRC)
|
| Lab |
IFX
|
| Street address |
9704 Medical Center Dr
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL9098 |
| Series (1) |
| GSE19071 |
Isolation and characterization of environmental Bacillus isolates which may harbor B. anthracis virulence genes |
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