Visualising data for Cancer referrals¶
Visualising Small variants¶
For visualisation of small variants, users should load the CRAM file(s) for their referral of interest. The colour and intensity of the reads and positions within a read indicates the sequence and quality of the alignment and sequence. Reads in regions with poor mapping quality are shown in white (or lighter colours). Base quality within a read is indicated by the brightness of the colour for an individual base, with brighter colours indicating better quality.
An example of a good quality small variant is shown in Figure 13. In this example, the reads in pink represent the calls on the forward strand, and those in blue the reverse strand. The variant in this example is supported by both strands with high base quality and there is little sequencing 'noise' around the area.
Visualising Copy Number Variants¶
Copy number variants (CNVs) can be visualised using either alignment (CRAM) files or coverage profiles (bigwig files), using either IGV.js or the desktop version of IGV. For large CNVs, visualising the coverage profiles (BigWig files) is recommended, since the genomic region of interest may be too large to load from a CRAM file. An example visualisation for coverage profiles is shown in Figure 14, showing the coverage for both germline (top) and tumour (bottom) samples. Here, a large deletion in the tumour sample is evident as a reduction in coverage.
For additional confirmation of CNVs, and to define the breakpoints more precisely, it is beneficial to review supporting reads in the CRAM files. For large CNVs, it is recommended to zoom into to breakpoint regions for breakpoint review. Further information for reviewing breakpoint reads is described below (see Visualising Structural variants for a Cancer referral).
Visualising Structural variants¶
IGV can be used to review and interpret sequencing reads which support identification of structural variants. Supporting read types may be "paired reads" where the location or orientation of the two reads in a read pair indicate a rearrangement, or "split reads" where the breakpoint has been directly sequenced. Reviewing paired and split reads can be used to refine breakpoints for CNVs as well as to assess and interpret complex rearrangements.
Inversions¶
IGV can be used to visualise genomic inversions by selecting the option to colour alignments by pair orientation ("Colour alignments>by pair orientation"). Blue and teal reads indicate read pairs in which the orientation of the reads supports a segment of the genome being inverted. "Split reads" can be visualised by selecting the option to view soft-clipped bases. An example showing the breakpoints of an inversion is shown in Figure 15.
Translocations¶
Translocations can be visualised by selecting the option to colour alignments by insert size ("Colour alignments>by insert size") . In IGV, reads are coloured according to the chromosome on which the mapped paired-read is aligned. Reviewing split-reads can also be helpful in assessing the quality and refining the precise breakpoints for translocation events. An example of a translocation can be found in Figure 16, showing a translocation between chromosome 12 and chromosome 21, where the reads aligned on chr12 are coloured yellow, indicating that the paired-read is aligned to chr21, and the reads aligned on chr21 are coloured purple indicating that the paired-read is aligned to chr12.
Further guidance for reviewing and interpreting signatures of copy number and structural variants from CRAM files can be found https://software.broadinstitute.org/software/igv/interpreting_insert_size and https://software.broadinstitute.org/software/igv/interpreting_pair_orientations.