Phase complex mutations in e.g. cancer

How haplotypes can be phased with Xdrop™ and single molecule sequencing

Knowing the chromosomal phase of genomic sequence variants is important for genetic analysis in many diseases like cancer.

The recognition of haplotype phasing as an important tool for better development of personalized treatments and precision medicine is currently increasing.

Standard genotyping arrays, Sanger sequencing and short-read sequencing (e.g. Illumina NGS), commonly used in cancer research, identify the mutations present without revealing their arrangement as haplotypes. Xdrop™ enrichment followed by single molecule long-read sequencing enables researchers to resolve haplotype phasing in human cancer samples easier and quicker than current methods. This has been demonstrated by researchers at the SciLifeLab, Uppsala University and Cancer Center at Karolinska Institute in collaboration with Samplix.

Briefly explained, five different primer sets were designed with the Samplix online design tool to amplify different ~200 bp regions distributed across a 70 Kb region on chromosome 17p13 encompassing the entire TP53 gene.

After the Xdrop™ dPCR enrichment and dMDA amplification steps, the harvested DNA was subjected to SMRTbell library preparation followed by PacBio long read sequencing. Due to the enrichment, the region was deeply sequenced, and high accuracy consensus reads where aligned. The high fidelity of consensus sequence allowed the haplotype phasing of three mutations located several hundred nucleotides apart (see figure A below). Phasing of three pathogenic mutations; c.913A>T (nonsense), c.673-2A>T (splice site), c.701A>G (missense) and normal wildtype (WT) revealed that none of the point mutations occurred in the same consensus read, meaning the mutations were present on separate alleles in the CLL patient cell population.

Poster after Ida Höijer A0 graf

Figure A. Phasing of three pathogenic mutations;
913A>T (nonsense), 673-2A>T (splice site), 701A>G (missense) and normal wildtype (WT) in a single CLL patient sample.

Each of the grey lines shows a consensus read. A mutation is indicated with a colored line across this. Red boxes indicate locations of the three mutations. As seen in the data and schematically drawn, none of the point mutations occur in the same consensus read, meaning they are present on separate alleles in the CLL patient cell population.

Do you want to know more? 

Download the TP53 poster for more details on this project.

Poster after Ida Höijer A0