Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data
- Luisa A. Marcelino*,
- Vadim Backman†,
- Andres Donaldson*,
- Claudia Steadman*,
- Janelle R. Thompson*,‡,
- Sarah Pacocha Preheim*,
- Cynthia Lien*,
- Eelin Lim§,
- Daniele Veneziano*, and
- Martin F. Polz*,¶
- *Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139;
- †Biomedical Engineering Department, Northwestern University, Evanston, IL 60208; and
- §Department of Biology, Temple University, Philadelphia, PA 19122
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Edited by J. Craig Venter, The J. Craig Venter Institute, Rockville, MD, and approved June 13, 2006 (received for review February 22, 2006)
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Fig. 1.Performance of the analytical cross-hybridization predictor. (a) Comparison of cross-hybridization probability obtained by analytical predictor (Eq. 4) with experimental data obtained by spike-in microarray experiments where total RNA of five different bacteria (V. cholerae, Vibrio anguillarum, Vibrio vulnificus, Vibrio alginolyticus, and Escherichia coli) were mixed individually with artificial RNA community samples at different amounts. (b) Comparison of ΔGjk―/ΔGjj ratios calculated by the analytical predictor (Eq. 5) with the ΔGjk―/ΔGjj ratios calculated by mfold, as a function of sequence identity. Error bars are standard errors.
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Fig. 2.V. cholerae (V.C.) signal within artificial communities before (a–c) and after (d–f) application of the algorithm (Eqs. 1 and 2). Artificial RNA communities contained 12.5, 1.25, and 0.5 μg of eukaryal RNA with 6.25, 1.25, and 0.25 ng of V. cholerae (black bar) RNA (between 0.1% and 0.05% of the total RNA). RNA abundances were determined by either averaging normalized background-subtracted intensities over all of the probe sets targeting bacteria-specific RNA after outlier removal (a–c) or by application of Eqs. 1 and 2 (d–f).
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Fig. 3.Quantification of spiked V. cholerae RNA in artificial and natural RNA communities (Eqs. 1–5). RNA abundances returned by the algorithm (Eqs. 1 and 2) show Langmuir dependence on true abundances (Eq. 3) with linear (0.25 ng ≤ true RNA abundance ≤20 ng) (a), nonlinear (20 ng < true RNA abundance ≤60 ng) (b), and plateau regimes (>60 ng).
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Fig. 4.Identification of seven naturally occurring closely related Vibrio taxa (16S rRNA sequence identity >95%) in coastal seawater samples (Eqs. 1–5). Relative RNA abundances (percent of total RNA), ranging from 0.005% to 0.15%, and absolute RNA abundances (nanograms of RNA in hybridized sample), ranging from 0.12 to 22.50 ng, are indicated for some Vibrio populations.
Footnotes
- ¶To whom correspondence should be addressed. E-mail: mpolz{at}mit.edu
- © 2006 by The National Academy of Sciences of the USA
