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Each array was normalised individually, followed by normalisation across all arrays to allow comparison.
Normalisation across arrays was performed initially using a variety of strategies: (a) do-nothing (b) separate quantile normalisation of the U and M channels and recomputation of β-values, (c) quantile normalisation of β-values, (d) quantile normalisation followed by adjustment for batch, DNA input and BSC efficiency effects, and (e) adjustment for batch, DNA input and BSC efficiency effects.
Initial data analysis was performed using GenomeStudio version 2010.3 (Illumina), using average normalisation across all the samples.
Normalisation across all arrays was performed on a log scale using the quantile normalisation method [ 45] implemented in lumi normalisation package [ 46].
We ameliorated this through QC and normalisation across plates, and then jointly clustering KIR3DL1 and KIR3DS1, to exploit the correlation between the ΔCt values.
We downloaded background-corrected gene expression values from the Gene Expression Omnibus (GEO) database (accession number GSE6536), and then carried out quantile normalisation across replicates of a single individual and subsequently median normalisation across all individuals by using the R package beadarray [ 13].
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5506 datapoints (spots) passing the normalisation filters across all arrays (18 datasets) were utilised for this purpose.
The remainder were normalised within each array using printtiploess, and subsequently normalised across arrays using quantile normalisation.
Prior to assessing differential expression, data were normalised across libraries using the trimmed mean of M values normalisation method [ 25].
Each array was globally normalised; global normalisation was used as it ensures that the measured intensities are comparable across all slides.
Data normalisation was performed across all arrays using quantile normalisation.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com