Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

5.2 Array Comparative Genomic Hybridization Methods
which transcript it was extracted. If the cut site was less than 17nt from the
end of the transcript, we extended the sequence with adenine stretches, rep-
resenting the poly-adenine (poly-A) tail. In these cases, additional upstream
tags were also extracted until one tag fully contained in the Ensembl cDNA
sequence was obtained. We disregarded Ensembl transcripts not belonging
to a gene of biotype "protein_coding" or "processed_transcript". Ensembl
annotates genes of several other biotypes, e.g. pseudogenes and small RNAs,
but those annotations are not based on full-length transcript sequences, so
we would not expect to find valid virtual tags in those transcripts. For the
majority of this study, we used a conservative subset of the virtual tags from
SAGE Genie and Ensembl comprising 25,593 unique tags assigned to 15,103
genes (Table 5.2). Specifically, we used SAGE Genie tags extracted from to the
3’-most cut site in RefSeq or MGC cDNAs having a poly-A tail or a polyadeny-
lation signal, and Ensembl tags from transcripts of type "protein_coding" or
"non_coding". Any virtual tags that mapped to multiple loci by these criteria
were excluded. For certain analyses, we made use of more comprehensive vir-
tual tag sets. In addition, we determined unique, perfect matches for tags to
the genome using Bowtie as described above. We calculated a single expression
value for each gene in each cell line by summing the counts of tags assigned to
the gene.
5.2 Array Comparative Genomic Hybridization
We re-analysed the array comparative genomic hybridization (CGH) data de-
scribed by Pollard et al [404] CGH was performed with Human Genome CGH
Microarray 4x44K arrays (Agilent), using genomic DNA from each cell line
hybridised in duplicate (dye swap) and normal human female DNA as ref-
erence (Promega). Log2 ratios were computed from processed Cy3 and Cy5
intensities reported by the software CGH Analytics (Agilent). We corrected
for effects related to GC content and restriction fragment size using a modi-
fied version of the waves array CGH correction algorithm [271]. Log2 ratios
were adjusted by sequential loess normalization on three factors: fragment GC
content, fragment size, and probe GC content. These were selected after in-
vestigating dependence of log ratio on multiple factors, including GC content
in windows of up to 500 kilobases centred around each probe. The Biocon-
ductor package CGHnormaliter [506] was then used to correct for intensity
dependence and log2 ratios scaled to be comparable between arrays using the
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