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SYGNIS presents feasibility data for cell free DNA amplification with TruePrime™ technology at two recent meetings

SYGNIS has presented data showing feasibility of a TruePrime™-mediated amplification of so-called cell free DNA in the meeting “10th ISMRC International Symposium on Minimal Residual Cancer: Liquid Biopsy in Cancer Diagnostics and Treatment” in March in Hamburg, Germany, and at the at the CHI’s Fourth International Molecular Diagnostics Europe in Lisbon, Portugal. The data were presented in poster format entitled: “TruePrime™ liquid biopsy: Tuning whole genome amplification towards improving the sensitivity of circulating tumor DNA analysis” and authored by Angel Picher and colleagues.

Low bias cfDNA amplification with TruePrime™ technology

We have already talked about the concept of liquid biopsy, and the great opportunities that arise from this concept in a previous post (from March 7 2016). One problem in many plasma samples is that the amounts of DNA that can be extracted is very low and this poses difficulties for subsequent analyses. SYGNIS did present data from plasma of healthy donors which showed that pretreatment and TruePrime™-amplification led to µg amounts of DNA from 1 ng down to 100 fg’s. The amplificates could be successfully subjected to whole genome sequencing with Illumina technology, and mapped to the human genome (Figure 1). The evenness of coverage of different chromosomes, detectability of all chromosomal regions, and the detectable read depth reduction over the X and Y chromosomes (the DNA was from a male – 22 diploid autosomes and one X and Y chromosome, respectively) all suggest that the amplification bias was surprisingly low.

 

Figure 1: Circos plot depicting read coverage over the human genome. The sample was obtained by amplifying 100 pg of cell free DNA from healthy donors, and sequencing (Illumina HiSeq, paired end reads, 5 million read pairs were sequenced). Blue histogram bands show read depth at the respective position in the genome.

Accurate cfDNA SNVs detection with TruePrime™ technology

Furthermore, we used a targeted amplicon approach to obtain feasibility data from a cancer case. The case was an undifferentiated colorectal adenocarcinoma (T4a M1a L1 R1 G4). We amplified 36 pg of plasma DNA (quantified by Qubit) and compared this to non-amplified DNA extracted from a tumor biopsy. The AmpliSeq Cancer HotSpot panel v2 was used with 207 amplicons. DNA was PCR amplified, and sequenced on an Ion Proton sequencer. SNVs detected in the amplified cfDNA sample showed a stunning overlap with the tumor biopsy sample, both in presence of SNVs as well as in the allele frequency (Figure 2). This suggests that low bias was introduced during the amplification process, and that allele frequencies were well preserved. Although one SNV present in the tumor sample at app. 10% frequency was not detected in the amplified cfDNA, a number of SNVs were only found in the amplified cfDNA, one of them an activating KRAS A59S mutation that had not been diagnosed in the patient before.

Figure 2: Diagram showing SNVs identified for a colon cancer biopsy sample (“tumor tissue”) and a TruePrime™-amplififed cell free DNA sample (“amplified cfDNA B”). Allele frequencies are color-coded. Shown on the left are the positions of the SNVs. Analysis was done with the AmpliSeq Cancer HotSpot panel v2 with 207 amplicons covering key cancer mutations.

These data underscore the feasibility of SYGNIS’ approach for tuning TruePrime™ into a tool that can increase the sensitivity of cfDNA analyses and help to make some of the promises of liquid biopsy come true.