Analyse des GC-reichen TERT Promoters und des MGMT Methylierungsstatus in liquid biopsies - ein optimiertes Workflow für die Genomamplifikation von Einzelzellen und zellfreier DNA

Liquid biopsies have shown promising results. However, due to low DNA quantity and quality, the methodology needs to be optimized for single cells and cell-free DNA while taking into account the necessities of neuro-oncological molecular pathology, i.e. analysis of GC-rich regions and epigenetic markers. Therefore, we aimed to optimize the Ampli1 Whole Genome Amplification Kit for better amplification of GC-rich regions, combining the method"s advantages of high fidelity, excellent genome coverage and low allelic dropouts with the analysis of GC-rich regions in single cells. Furthermore, we included methylation-sensitive digestion for epigenetic analyses and adapted this method for the use with cell-free DNA.

DMSO and betaine were tested for their improvement in amplification of GC-rich DNA fragments in the Ampli1 WGA protocol. Successful amplification of GC-rich fragments was evaluated by PCR with fragments up to 70% GC and amplicons up to 85% GC. The WGA protocol was modified to allow for addition of the methylation-sensitive Hin6I enzyme in the digestion step. For the adaptation of the protocol to cell-free DNA, an end-repair and dA-tailing step was tested prior to the ligation of a modified adapter to the short cell-free DNA fragments and their subsequent amplification.

Conventional Ampli1 WGA failed to amplify DNA fragments above 60% GC content, while additives enabled the amplification of fragments up to 70% GC and the analysis of amplicons up to 80% GC. The inclusion of methylation-sensitive Hin6I digestion prior to adapter ligation and amplification allows for the analysis of ~ 300K CpG dinucleotides and 72K of the CpG dinucleotides in the Illumina EPIC 850K chip. MGMT methylation status was correctly analyzed in 100% of analyzed single GBM cells and PBLCs. Successful adaptation of the method for cell-free DNA by inclusion of DNA end repair and dA tailing resulted in ~ 2 µg of DNA for further analysis with 1 ng of input.

While existing methods for WGA of single cells perform well for the majority of assays, molecular analysis of certain neuro-oncological alterations in GC-rich regions (TERT promoter) and epigenetic changes are not feasible. With the adaptation of existing methodology, these analyses are possible on both single cells and cell-free DNA. Inclusion of betaine and methylation-sensitive enzymes enable the evaluation of methylation status of up to 300K CpG dinucleotides and genetic analysis of GC-rich regions by PCR and sequencing.