origin: monitoring well from TCE contaminated site site: B sampling campaign: 4 well: P1 treatment: after ERD (enhanced reductive dechlorination) sample type: environmental sample
Growth protocol
Groundwater samples were collected by SITA Remediation Company (Lyon, France) from four contaminated sites (B, F, G and H) by direct pumping in four monitoring wells per site (P1, P2, P3 and P4) drilled in the deep aquifer. P1: well located upstream to the contamination source. P2: well in the contamination source. P3 and P4: wells located downstream to the contamination source. For site B, the monitoring of ERD demonstration was performed through a total of 5 sampling campaigns: C1 (T=0), C2 (T=104 days), C3 (T=231 days), C4 (T=291 days) and C5 (T=378 days). For the three other sites (F, G and H), only one sampling campaign was performed after the treatment.
Extracted molecule
genomic DNA
Extraction protocol
Groundwater samples were transferred to the laboratory, filtered under vacuum and collected onto a 47 mm 0.2 µm pore size GTTP filter (Millipore). Filters were then cut in pieces and placed into 900 µL of nucleic extraction buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8.0) as described by Vetriani (Vetriani, C., Tran, H.V. and Kerkhof, L.J. (2003) Fingerprinting Microbial Assemblages from the Oxic/Anoxic Chemocline of the Black Sea. Appl. Environ. Microbiol., 69, 6481-6488). In this protocol, freeze-thaw cycles were replaced by beat betting 1 min at 30 Hz with 1 g of glass beads of ≤106 μm in diameter (Sigma-Aldrich). gDNA were extracted using a standard phenol-chloroform-isoamyl alcohol (25:24:1) method, followed by a second extraction with an equal volume of chloroform. Nucleic acids were then precipitated, washed in 70% ethanol and resuspended in nuclease-free water.
Label
Alexa5
Label protocol
The BioPrime Total Genomic Labelling System (Invitrogen, Carlsbad, California, USA) was used to label the gDNA with Alexa Fluor 3 or 5 fluorescent dyes. The reactions were performed from 1 µg of gDNA template following the manufacturer's instructions and allowed an increase of at least 6-fold the amount of starting gDNA material.
Hybridization protocol
For each hybridization experiment, labeled gDNA were vacuum dried and resuspended in the NimbleGen Hybridization Buffer. The arrays were hybridized on a 4-bay NimbleGen Hybridization System (Roche NimbleGen) at 42°C for 72 hr. Arrays were washed with NimbleGen wash buffers I, II and III according to vendor protocols.
Scan protocol
Arrays were scanned using a Scanner Innoscan 900AL (Innopsys, Carbonne, France) at 2 μm resolution. Individual array images were acquired independently, adjusting the PMT gain for each image as recommended using the software Mapix® (Innopsys). Then, for each array image, raw expression data was extracted using the NimbleScan software v2.5. (Roche NimbleGen) and feature intensities were exported as .pair files.
Description
Complex environment. gDNA extracted from contaminated site B, campaign C4 in the well P1.
Data processing
The background noise was determined using random probes present on the microarrays (5,707 probes in our experiment). This background noise is defined by two components: the background median intensity (Bposition) and its dispersion (Bdispersion). Finally, a modified signal-to-noise ratio called SNR’ and based on the formula of Verdik (Verdick, D., Handran, S. and Pickett, S. (2002) In LLC, D. P. (ed.), In G. Kamberova (ed.), DNA array image analysis: nuts and bolts. Salem, MA., pp. p. 83-98) is calculated as follows in order to reduce-centralize our data: SNR’ = (probe signal intensity - Bposition)/ Bdispersion. However, spatial effect across array surface is a predominant within-slide experimental artifact that needs to be eliminated before any other normalization procedure (Wang, X., He, H., Li, L., Chen, R., Deng, X.W. and Li, S. (2006) NMPP: a user-customized NimbleGen microarray data processing pipeline. Bioinformatics, 22, 2955-2957). Thus, for all array images obtained in this work, surface was segmented to 16 sub-squares according to probe position (X, Y) indicated in pair report. A Perl script was developed to calculate local background noise in all sub-squares and the median SNR’ retrieved from the eight replicates of each probe. Finally, another Perl script was implemented to summarize each replicate probe treated and determine the median value between the eight replicates. Positive hybridization was considered significant for probes having an SNR’ > 1.5 (value to avoid all false positives).
