Investigation Title Genotyping of twenty Pseudomonas aeruginosa strains Comment[Submitted Name] Ausubel P. aeruginosa Genotyping Experimental Design genotyping_design genotyping by array Experimental Design Term Source REF EFO Comment[ArrayExpressReleaseDate] 2006-10-06 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-MEXP-824 Comment[MAGETAB TimeStamp_Version] 2011-06-28 22:30:01 Last Changed Rev: 14857 Experimental Factor Name strain Experimental Factor Type strain_or_line Experimental Factor Term Source REF Person Last Name Rahme Kucherlapati Urbach Montgomery Lee Grills Feinbaum Miyata Diggins Wu Ausubel Liberati Deziel Saucier He Li Friedman Person First Name Laurence Raju Jonathan Kate Daniel George Rhonda Sachiko Lenard Gang Frederick Nicole Eric Maude Jianxin Li Lisa Person Mid Initials G M Person Email danlee@molbio.mgh.harvard.edu Person Phone 617-726-5950 Person Fax Person Address 185 Cambridge St., CPZN7250, Boston, MA, 02114, USA Person Affiliation Molecular Biology, Massachusetts General Hospital Harvard Medical School - Partners Healthcare Cente Molecular Biology, Massachusetts General Hospital Harvard Medical School - Partners Healthcare Cente Molecular Biology Harvard Medical School - Partners Healthcare Cente Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Envivo Pharmaceuticals, Inc. Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Molecular Biology, Massachusetts General Hospital Harvard Medical School - Partners Healthcare Cente Harvard Medical School 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 2006-10-06 PubMed ID 17038190 Publication DOI 10.1186/gb-2006-7-10-r90 Publication Author List Lee DG, Urbach JM, Wu G, Liberati NT, Feinbaum RL, Miyata S, Diggins LT, He J, Saucier M, Deziel E, Friedman L, Li L, Grills G, Montgomery K, Kucherlapati R, Rahme LG, Ausubel FM. Publication Title Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Publication Status journal_article Publication Status Term Source REF Experiment Description 20 P. aeruginosa strains were genotyped using a custom spotted oligo array (Ausubel P. aruginosa genotyping 4.8K v1), which included primarily oligos for genes present in strain PA14 but absent in PAO1, and oligos for genes present in PAO1 but absent in PA14 (as well as controls). Protocol Name P-MEXP-30055 P-MEXP-30058 P-MEXP-30061 P-MEXP-30060 P-MEXP-30062 P-MEXP-30092 P-MEXP-30139 Protocol Type grow nucleic_acid_extraction labeling labeling hybridization feature_extraction bioassay_data_transformation Protocol Description Bacterial strains were grown in LB broth at 37 degrees Celcius for 16 hours. Each strain was subcultured 1:25 and 1:100 in LB and grown for an additional 2-3 hours at 37 degrees. OD(600) measurements were taken, and the equivalent of 1 ml at OD(600)=1.0 were collected. PA14, PAO1 and 18 additional P. aeruginosa strains described previously [Wolfgang MC, Kulasekara BR, Liang X, Boyd D, Wu K, Yang Q, Miyada CG, Lory S: Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2003, 100(14):8484-8489] was prepared from the equivalent of OD(600)=1.0 of starting material using the FastDNA kit (Bio101, part # 6540-400) according to the manufacturer's recommendations for bacterial chromosomal DNA. 11 ug of purified genomic DNA was digested with HaeIII as described [http://www.microarrays.org/protocols.html]. 2 ug of digested genomic DNA was directly labeled using the MICROMAX ASAP labeling kit (PerkinElmer, part #MPS544001KT) with Cy5. 2 ug of digested genomic DNA was directly labeled using the MICROMAX ASAP labeling kit (PerkinElmer, part #MPS544001KT) with Cy3. 2 ug of Cy3 labeled sample was mixed with 2 ug of Cy5 labeled sample.
80 ug of polyA, 40 ug of salmon sperm DNA, and 100 ug of were added to the sample, along with H2O to a final volume of 65 ul.
The sample were heated to 95 degrees for 3 minutes, and combined with 65 ul of 2X hyb buffer (6X SSC, 50 mM Hepes pH 7.0, 50% formamide) and injected into the hybridization chamber of a GeneTAC Hybridization Station.
Hybridiztions were performed for 16 hours at 44 degrees, followed by the following washes:
2 washes with 2X SSC / 0.1% triton at 30 degrees
3 washes with 2X SSC at 25 degrees
2 washes with 0.2X SSC at 23 degrees
2 washes with 0.06X SSC at 23 degrees
Slides were removed from the hybridization chamber and washed 3 times (by dipping) in 0.06X SSC.
Slides were dried by placing them in a 50 ml conical tube (Falcon) and spinning at 800 RPM for 5 minutes with no brake.
Dried arrays were scanned with an Axon GenePix 4000B scanner using the GenePix Pro 3.0 software, and preliminary data analysis was performed using the BioArray Software Environement (BASE) developed at the MGH Microarray Core Facility (https://base.mgh.harvard.edu/) as described in the Transformation protocols (Ausubel - P. aeruginosa Transformation1).

STEP 3 (OF 3): CONVERTING TO PRESENT/INDETERMINATE/ABSENT CALLS
We attempted to determine present or absent calls for each gene as described [Kim CC, Joyce EA, Chan K, Falkow S: Improved analytical methods for microarray-based genome-composition analysis. Genome Biol 2002, 3(11):RESEARCH0065]; however, our data set was too small with respect to the total number of genes assayed for this method to be effective. Instead, the distribution of log2 ratio values was plotted for each strain (using a bin size of 0.25), and a roughly bimodal distribution was observed in all cases, with one peak of log2 ratios corresponding to genes present in the tested strain (generally centered between 0 and -1) and a second, broad peak corresponding to absent genes (with log2 ratios of approximately -1.5 and below). For each strain, a range was determined that lay in between the two peaks, for which the present or absent call would be classified as indeterminate. Log2 ratio values above this range resulted present calls, and values below this range were absent calls.
Present calls were represented as 3.
Indederminate calls were represented as 2.
Absent calls were represented as 1.
The empirically determined ranges of log2 ratios for indeterminate values are as follows: -1.2 to -0.8 for PA14 and PAO1; -1.4 to -1.0 for CF27; -1.7 to -1.3 for 62, 6077, 19660, CF5, MSH3, MSH10, S54485, U2504, UDL and X13273; -2.0 to -1.6 for CF18, CF127, E2, JJ692, and PAK; -2.2 to -1.8 fro X24509; -2.8 to -2.4 for S35004. Using these cut-off values, oligos were identified that failed to yield the expected results with respect to PA14 and/or PAO1 (i.e. they were called present in PA14 when BLAST results predict that they should be absent). In addition to these oligos that failed to yield the expected results, oligos corresponding to negative controls were omitted form the final analysis and are marked as OMITTED in the Array Description File under the column header Reporter Comment. Oligos labeled "used" were inclued in the final analysis, and oligos labeled "used; intergenic" were included in the final analysis and corresponded to intergenic (non-coding) regions of the reference genome. Protocol Parameters max temperature;min temperature;stop time;start time; Extracted product;Amplification; Label used;Amount of nucleic acid labeled;Amplification; Label used;Amplification;Amount of nucleic acid labeled; Chamber type;Quantity of label target used;temperature;time;Volume; Protocol Hardware Protocol Software GenePix Pro [Axon Instruments] Protocol Contact Protocol Term Source REF SDRF File E-MEXP-824.sdrf.txt Term Source Name mo ArrayExpress EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ Term Source Version