Abstract
Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues is becoming the standard for identifying the best treatment regimen. However, about 25% of all samples are rejected for genetic analyses for reasons that include too little tissue to extract enough high quality DNA. One way to overcome this is to do whole-genome amplification (WGA) in clinical samples, but only limited studies have tested different WGA methods in FFPE cancer specimens using targeted next-generation sequencing (NGS). We therefore tested the two most commonly used WGA methods, multiple displacement amplification (MDA-Qiagen REPLI-g kit) and the hybrid or modified PCR-based method (Sigma/Rubicon Genomics Inc. GenomePlex kit) in FFPE normal and tumor tissue specimens. For the normalized copy number analysis, the FFPE process caused none or very minimal bias. Variations in copy number were minimal in samples amplified using the GenomePlex kit, but they were statistically significantly higher in samples amplified using the REPLI-g kit. The pattern was similar for variant allele frequencies across the samples, which was minimal for the GenomePlex kit but highly variable for the REPLI-g kit. These findings suggest that each WGA method should be tested thoroughly before using it for clinical cancer samples.
Highlights
The advent of the polymerase chain reaction (PCR) for genetic analyses[1,2,3] has led to significant progress in several fields, including forensic science, drug selection, and molecular diagnoses of diseases such as cancer[4,5,6,7,8]
It is important that different whole genome amplification (WGA) methods using the targeted next-generation sequencing (NGS) panels should be evaluated in clinically challenging samples like FFPE DNA
When we broke down the analysis by tissue type and WGA kit, we observed that NGS libraries prepared with either REPLI-g or GenomePlex had significantly lower coverage uniformity than non-WGA samples regardless of the tissue type (Figure S1a)
Summary
The advent of the polymerase chain reaction (PCR) for genetic analyses[1,2,3] has led to significant progress in several fields, including forensic science, drug selection, and molecular diagnoses of diseases such as cancer[4,5,6,7,8] Most of these PCR-based applications require at least nanogram level of DNA to robustly amplify and analyze original DNA9–11, which is not available from every biological or clinical sample. The best examples are Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and Rubicon Genomics Inc.’s technologies, such as PicoPLEX and GenomePlex (Sigma-Aldrich)[9, 10, 16] These hybrid methods increase the amplification uniformity while keeping reasonably low false positive and negative rates[10]. We evaluated the two most commonly used WGA methods, MDA and hybrid amplification[10], on challenging clinical specimens using a targeted NGS cancer panel with selected genes. The two WGA methods were thoroughly evaluated for DNA yield and quality, library prep result, and the final sequencing status using targeted NGS panels
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