Mutation Detection with NGS

Molecule Tutorials - Herong's Tutorial Examples

∟Mutation Detection with NGS

Provides a tutorial example on how to use NGS (Next-Generation Sequencing) technology to detect genomic mutations.

After understanding the NGS (Next-Generation Sequencing) technology, let's see how NGS can be used to detect genomic mutations from patient's samples.

A doctor wants to verify if a tumor patient has any mutations at the position of 600 of the protein sequence expressed by the BRAF gene.

So 2 tissue samples, normal tissue and tumor tissue, are sent out separately as NGS tests. The raw data of reads (aligned nucleotide letter strings) from both tests are listed on the left of the diagram (source: ncbi.nlm.nih.gov) below:

On the right of the above diagram, a detailed analysis of the NGS reads is also provided by the source on how a BRAF V600E mutation is detected.

But you also read my analysis listed below:

Top section shows reads from tumor tissue.
Bottom section shows reads from tumor tissue.
Yellow lines represent forward reads aligned to the reference genome.
Blue lines represent backward reads aligned to the reference genome.
Horizontally, all lines are aligned precisely to show the same section of the patient's genome sequence.
The center of each line is aligned to the position of 140,453,136 (part of the BRAF gene) in chromosome pair 7, which has nucleotide letter T for Thymine.
All reads match perfectly with the reference genome, except those red dots at the center of some lines. The red dot represents nucleotide letter A for Adenine instead of T from the reference genome.
Lines with red dots in the Tumour tissue section counts for 50% of the total reads. This suggests that a point mutation occurred on only 1 of the 2 paired chromosomes, which is called heterozygous.
The mutated BRAF genes will be expressed as mutated BRAF proteins at position 600 with Valine (V) being replaced by Glutamic Acid (E), which is noted as "BRAF:p.V600E".
In the Normal Tissue section, there is only 1 red dot at the center of all reads. It will be discarded as error. This suggests that no mutations found in normal tissue. So the mutation found in tumor tissue is not inherited patient's parents.