Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

6.2 Tag mapping Results
Figure 6.2: Step by step diagram of the extraction, filtering and mapping phases
for reads and tags. The extracted tag population is coloured with different shades of
grey that represent a different origin (adapter, mitochondrial or ribosomal tag). In
the mapping phase the diagram to the right represents the human reference genome
and some of the filtered tags (light grey) are mapping onto some known transcripts
(gene A, gene B) and non-coding regions.
the reference genome (adapter tags formed by the ligation of two sequencing
primers, and mitochondrial RNA), as well as rRNA sequences, were filtered
out. As shown in figure 6.3, on average more than 90% of tags remained un-
filtered, meaning that most of the sequenced data were available for us to use
in subsequent analyses, the first one being alignment to the reference genome.
Finally, the tag sequences contained within the recounted and filtered reads
were mapped in two complementary ways. Notice that, in order to maximise
the ability of the aligner to effectively map our short tag sequences to the ref-
erence genome, we included the CATG recognition site of the MmeI anchoring
enzyme, at the 5’ of every tag sequence; this generated 21nt tag sequences that
were used for alignment to the reference genome. For each library, we mapped
the so-generated pool of 21nt tags to the human reference genome, as well as
to a virtual tag-to-gene library that we assembled from already existing tag-to-
gene libraries and a complementary one that we generated programmatically.
The tag mapping strategy we adopted was a hierarchical one, as summarised in
figure 6.4. This allowed us to generate sets of decreasing stringency - regarding
the number of tags mapping to known transcripts - that were best fit for differ-
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