Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

5.6 Differential Isoform Expression Methods
Category 4. Is there no literature implicating the gene in any cancer?
If W = total weighted index and C = cancer index, then if W > 10 (internal
parameter, does not appear in fig 5.6), the gene is assigned to category one; if 1
< W < 10, the gene is assigned to category two; if W = 0 and C > 10 (internal
parameter, does not appear in fig 5.6) then the gene is assigned to category
three; finally, if W = 0 and C = 0 then the gene is assigned to category four.
5.6 Differential Isoform Expression
With the aim of establishing Tag-seq as a sensitive technique for the iden-
tification of 3'UTR transcript isoforms that are differentially expressed be-
tween GNS and NS cell lines, we evaluated the differential expression of 4,727
genes with multiple expressed tags using two alternative methods: the non-
parametric χ 2 test, and the logarithmic algorithm adapted from Morrissy et
al [346]. Non-parametric methods are applied when the assumptions for para-
metric methods about the underlying distribution of the data are not met,
namely that the data is normally distributed or that sample size is large enough
to support the assessment of the normality distribution. Whilst the normal-
ity assumption is satisfied when tested against the 28,351 tags mapping over
16,025 genes in the six libraries altogether, sample size drops to a range of two
to 14 tags per gene when assessing differential expression over a single tran-
script isoform. With a sample size smaller than 30, the use of non-parametric
methods is often advisable. Due to the nature of the SAGE protocol, tags map
at the 3'UTR of a gene and identify potential splicing isoforms. We conducted
a χ 2 test with the chisq.test function in R on all tags mapping on to the same
gene of the 12,794 tags expressed at higher than 10 tpm in at least one GNS
and one NS library.
In the second approach, following the logarithmic ratio method adapted by
Morrissy et al [346], the isoform expression between the GNS and NS cell lines
was evaluated by analyzing the expression of tags that mapped either twice on
the same gene or, if more than twice, for all their pair combinations. Again,
only tags with a minimum expression of 10 tpm in at least one GNS and one
NS library were considered (Table 5.3). The ratio of normalised expression
for each tag pair was calculated in each GNS and NS library, and multiplied
by the natural logarithm of the difference between the expression of each tag
in the pair, ensuring that the more highly expressed tag pairs would also be
more highly ranked (equations 5.4 and 5.5). For pairs of genes with positive
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