Investigation Title	Comparative genomic hybridization of 38 Bartonella henselae isolates from natural feline populations and clinical strains to investigate genomic diversity and the effect of growth time 	
Comment[Submitted Name]	Whole-genome diversity in the natural population of feline and clinical Bartonella henselae strains	
Experimental Design	strain_or_line_design	comparative genomic hybridization by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]	2006-08-29	
Comment[AEMIAMESCORE]	5	
Comment[ArrayExpressAccession]	E-TABM-88	
Comment[MAGETAB TimeStamp_Version]	2011-01-23 21:21:05 Last Changed Rev: 14857	
Experimental Factor Name	StrainOrLine	Time	
Experimental Factor Type	strain_or_line	time	
Experimental Factor Term Source REF	
Person Last Name	Lindroos	
Person First Name	Hillevi	
Person Mid Initials	
Person Email	Hillevi.Lindroos@ebc.uu.se	
Person Phone	
Person Fax	
Person Address	
Person Affiliation	Department of Molecular Evolution, Uppsala University, Sweden	
Person Roles	submitter	
Person Roles Term Source REF		
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2006-08-29	
PubMed ID	16936024	
Publication DOI	16936024	
Publication Author List	Lindroos, Hillevi; Vinnere, Olga; Mira, Alex; Repsilber, Dirk; Naslund, Kristina; Andersson, Siv G. E.	
Publication Title	Genome Rearrangements, Deletions, and Amplifications in the Natural Population of Bartonella henselae	
Publication Status	journal_article	
Publication Status Term Source REF	
Experiment Description	The genomic diversity of 38 Bartonella henselae isolates was studied by comparative genomic hybridizations. In addition, the effect of growth time (5 or 10 days) was studied for 5 strains.	
Protocol Name	P-MEXP-8545	P-TABM-354	P-MEXP-8547	P-MEXP-8549	P-MEXP-8550	P-TABM-361	P-TABM-362	P-TABM-358	P-TABM-359	P-MEXP-8551	P-TABM-364	P-TABM-363	P-TABM-355	P-TABM-365	P-TABM-366	
Protocol Type	grow	grow	nucleic_acid_extraction	labeling	labeling	labeling	hybridization	hybridization	hybridization	hybridization	image_acquisition	image_acquisition	bioassay_data_transformation	bioassay_data_transformation	bioassay_data_transformation	
Protocol Description	Bacteria were grown on blood agar plates containing 5% horse blood in a 5% CO2-enriched atmosphere.	Bacteria were grown on blood agar plates containing 5% horse blood in a 5% CO2-enriched atmosphere.	Bacteria from one agar plate were collected with the help of a sterile cotton applicator and suspended in 500 ul PBS. After centrifugation, DNA was isolated with the AquaPure DNA Isolation Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturers instructions. Precipitated DNA was resuspended in 100 ul DNA Hydration Solution (AquaPure Isolation Kit).	2 ug genomic DNA in a total volume of 7.7 ul water was mixed with 8 ul 2.5 X random primers/reaction mix (BioPrime DNA labeling kit, Invitrogen), boiled for 5 minutes and placed on ice. The primed DNA was mixed with 0.8 ul 25 x Nucleotide mix (12.5 mM dATP, dCTP, dGTP; 5 mM dTTP) and 2 ul 1 mM Cy3-dUTP (Amersham Biosciences) and 0.5 ul Klenow enzyme (BioPrime DNA labeling kit, Invitrogen). The sample was incubated for 1 hour at 37 C, and the reaction was stopped with 2 ul Stop Buffer (BioPrime DNA labeling kit, Invitrogen). Labeled samples to be co-hybridized were combined and cleaned with MinElute Reaction Cleanup Kit (Qiagen), and eluted in 10 ul Buffer EB.	2 ug genomic DNA in a total volume of 7.7 ul water was mixed with 8 ul 2.5 X random primers/reaction mix (BioPrime DNA labeling kit, Invitrogen), boiled for 5 minutes and placed on ice. The primed DNA was mixed with 0.8 ul 25 x Nucleotide mix (12.5 mM dATP, dCTP, dGTP; 5 mM dTTP) and 2 ul 1 mM Cy5-dUTP (Amersham Biosciences) and 0.5 ul Klenow enzyme (BioPrime DNA labeling kit, Invitrogen). The sample was incubated for 1 hour at 37 C, and the reaction was stopped with 2 ul Stop Buffer (BioPrime DNA labeling kit, Invitrogen). Labeled samples to be co-hybridized were combined and cleaned with MinElute Reaction Cleanup Kit (Qiagen), and eluted in 10 ul Buffer EB.	1 µg genomic DNA in a total volume of 7.7 µl water was mixed with 8 µl 2.5 X random primers/reaction mix (BioPrime DNA labeling kit, Invitrogen), boiled for 5 minutes and placed on ice. The primed DNA was mixed with 0.8 µl 25 x Nucleotide mix (12.5 mM dATP, dCTP, dGTP; 5 mM dTTP) and 2 µl 1 mM Cy3-dUTP or Cy5-dUTP (Amersham Biosciences) and 0.5 µl Klenow enzyme (BioPrime DNA labeling kit, Invitrogen). The sample was incubated for 1 hour at 37 C, and the reaction was stopped with 2 µl Stop Buffer (BioPrime DNA labeling kit, Invitrogen). Labeled samples to be co-hybridized were combined and cleaned with MinElute Reaction Cleanup Kit (Qiagen), and eluted in 10 µl Buffer EB.	Slides were pre-hybridized in 5X SSC, 0.1% SDS, 0.1 mg/ml BSA at 50 C for 30-60 minutes, then washed 2 times in 0.1X SSC at room temperature 5 minutes, and finally dipped in de-ionized water for 30 seconds and spinned dry with a slide centrifuge. The labelled DNA (Cy3 and Cy5, complete yield in 10 µl) was mixed with 50 µl (for LifterSlips) or 90 µl (for ordinary coverslips) hybridization solution (5X SSC, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA) and heated at 95 C 1 minute before application to the slide. Slides were hybridized for 12-15 hours at 50 C and washed by first dipping in 2X SSC, 0.1% SDS at 50 C until coverslip moved away, then kept in 2X SSC, 0.1% SDS at 50 C for 5 minutes, followed by 2 times 5 minutes in 0.1X SSC, 0.1% SDS at room temperature, then five times 1 minute in 0.1X SSC at room temperature, and finally rinsed in 0.01X SSC 5-10 seconds and spinned dry with a slide centrifuge.	Slides were pre-hybridized in 3X SSC, 0.1% SDS, 0.1 mg/ml BSA at 50 C for 30-60 minutes, then dipped in water, dipped in isopropanol, and spinned dry with a slide centrifuge. The labelled DNA (Cy3 and Cy5, complete yield in 10 µl) was mixed with 50 µl (for LifterSlips) or 90 µl (for ordinary coverslips) hybridization solution (3X SSC, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA) and heated at 95 C 1 minute before application to the slide. Slides were hybridized for 12-15 hours at 50 C and washed by first dipping in 2X SSC, 0.1% SDS at 50 C until coverslip moved away, then kept in 2X SSC, 0.1% SDS at 50 C for 5 minutes, followed by 10 minutes in 0.1X SSC, 0.1% SDS at room temperature, then four times 1 minute in 0.1X SSC at room temperature, and finally rinsed in 0.01X SSC 5-10 seconds and spinned dry with a slide centrifuge.	Slide was UV-crosslinked at 350 mJ/cm2. Slides were pre-hybridized in 5X SSC, 0.1% SDS, 0.1 mg/ml BSA at 50 C for 30-60 minutes, then washed 2 times in 0.1X SSC at room temperature 5 minutes, and finally dipped in de-ionized water for 30 seconds and spinned dry with a slide centrifuge. The labelled DNA (Cy3 and Cy5, complete yield in 10 µl) was mixed with 50 µl (for LifterSlips) or 90 µl (for ordinary coverslips) hybridization solution (5X SSC, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA) and heated at 95 C 1 minute before application to the slide. Slides were hybridized for 12-15 hours at 50 C and washed by first dipping in 2X SSC, 0.1% SDS at 50 C until coverslip moved away, then kept in 2X SSC, 0.1% SDS at 50 C for 5 minutes, followed by 2 times 5 minutes in 0.1X SSC, 0.1% SDS at room temperature, then five times 1 minute in 0.1X SSC at room temperature, and finally rinsed in 0.01X SSC 5-10 seconds and spinned dry with a slide centrifuge.	The labeled DNA (Cy3 and Cy5, complete yield in 10 ul) was mixed with 90 ul hybridization solution (3X SSC, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA) and heated at 95 C 1 minute before application to the slide. Slides were hybridized for 15 hours at 50 C and washed in the dark by first dipping in 2X SSC and 0.1% SDS at 50 C until coverslip moved away, then kept in 2X SSC and 0.1% SDS at 50 C for 5 minutes, followed by 10 minutes in 0.1X SSC and 0.1% SDS at room temperature, then four times 1 minute in 0.1X SSC at room temperature, and finally rinsed in 0.01X SSC 5-10 seconds and spinned dry with a slide centrifuge.	Slides were scanned with a GenePix 4100A scanner using the GenePix Pro 5.1 program at a resolution of 10 µM. PMT was manually adjusted to balance intensities in the two channels while avoiding a high number of saturated spots. Quantification was done with circular features.	Slides were scanned with a ScanArray 4000 scanner (Packard BioChip Technologies/PerkinElmer Inc.) using the ScanArray Express (version 2.1) software (PackardElmer Inc.) at a resolution of 10 µM. PMT was manually adjusted to balance intensities in the two channels while avoiding a high number of saturated spots. Quantification of scanned images was also performed with ScanArray Express with settings diam 130 µm, max diam 120%, min diam 80%. Spots were automatically flagged as bad if spot mean <= 1.70 * background mean, or if spot mean <= 400 + background mean. Spots were also visually inspected and adjusted or flagged if necessary.  Ch1 was used denotes the reference (Bartonella henselae Houston1) strain and Ch2 for the test strain.	Data was analyzed with the statistics program R using in-house scripts. Ch1 denotes the channel for the reference strain. Spots meeting any of the following criteria were considered bad and removed from analysis: spots flagged as bad or not found during quantification; Ch1 spot median below 40 times the Ch1 background median ; more than 10 % saturated pixels in either channel; less than 95 % of spot pixels have Ch1 intensity higher than background intensity + one standard deviation; less than 90 % of spot pixels have Ch1 intensity higher than background intensity + two standard deviations; background-corrected Ch1 spot median intensity below 0.2 times the overall slide median Ch1 intensity; or the PCR-reaction used to generate the probe had failed (no visible band on gel). For each channel, median background intensities were subtracted from the median spot intensities, and the spot intensities were divided by the global mean background-corrected intensity. log2-ratios (M-values) computed as log2(Ch2/Ch1). Global normalization was performed by shifting the distribution of M-values such that the peak (identified by Gaussian kernel density estimation), corresponding to the majority of genes, was centered at M = 0. Plots of M vs average intensity (A = 0.5*(log2(Ch1)+log2(Ch2)) and M vs log2(Ch1) did not indicate any intensity-dependent dye bias lo loess normalization was not done.	Data was analyzed with the statistics program R using in-house scripts. Ch1 denotes the channel for the reference strain. Spots meeting any of the following criteria were considered bad and removed from analysis: spots flagged as bad or not found during quantification; Ch1 spot median below 5 times the Ch1 background median ; more than 10 % saturated pixels in either channel; less than 95 % of spot pixels have intensity higher than background intensity + one standard deviation; less than 90 % of spot pixels have intensity higher than background intensity + two standard deviations; background-corrected Ch1 spot median intensity below 0.2 times the overall slide median Ch1 intensity; or the PCR-reaction used to generate the probe had failed (no visible band on gel). For each channel, median background intensities were subtracted from the median spot intensities, and the spot intensities were divided by the global mean background-corrected intensity. log2-ratios (M-values) computed as log2(Ch2/Ch1). Global normalization was performed by shifting the distribution of M-values such that the peak (identified by Gaussian kernel density estimation), corresponding to the majority of genes, was centered at M = 0. Plots of M vs average intensity (A = 0.5*(log2(Ch1)+log2(Ch2)) and M vs log2(Ch1) did not indicate any intensity-dependent dye bias lo loess normalization was not done.	Data was analyzed with the statistics program R using in-house scripts. Ch1 denotes the channel for the reference strain. Spots meeting any of the following criteria were considered bad and removed from analysis: spots flagged as bad or not found during quantification; Ch1 spot median below 5 times the Ch1 background median ; more than 10 % saturated pixels in either channel; less than 95 % of spot pixels have intensity higher than background intensity + one standard deviation; less than 90 % of spot pixels have intensity higher than background intensity + two standard deviations; background-corrected Ch1 spot median intensity below 0.2 times the overall slide median Ch1 intensity; or the PCR-reaction used to generate the probe had failed (no visible band on gel). For each channel, median background intensities were subtracted from the median spot intensities, and the spot intensities were divided by the global mean background-corrected intensity. log2-ratios (M-values) computed as log2(Ch2/Ch1). Global normalization was performed by shifting the distribution of M-values such that the peak (identified by Gaussian kernel density estimation), corresponding to the majority of genes, was centered at M = 0. After inspection of plots of M vs average intensity (A = 0.5*(log2(Ch1)+log2(Ch2)) and M vs log2(Ch1), loess-correction was done on M vs Ch1 to correct for intensity-dependent dye bias.	
Protocol Parameters		growth time;	
Protocol Hardware	
Protocol Software	
Protocol Contact	
Protocol Term Source REF			mo	mo	mo					mo	
SDRF File	E-TABM-88.sdrf.txt	
Term Source Name	mo	The MGED Ontology	ArrayExpress	mo	EFO	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version