Somatic oncology research

• Somatic oncology research focuses on cancers resulting from DNA alterations and relies on the ability to find these alterations in DNA samples
• Due to the challenging process of collecting precious DNA samples, highly efficient sample preparation solutions are needed to unlock their research potential
• Roche sample prep solutions provide workflows that enable high sequencing efficiency for somatic oncology research applications

Somatic oncology research focuses on the study of cancers resulting from DNA alteration that occurs after conception; i.e., alteration that was not inherited. Two types of DNA alterations exist:

1. Genetic variation impacts the underlying gene structure (DNA sequence), e.g., (i) single-nucleotide polymorphisms (SNPs); (ii) small insertions and deletions (indels); and (iii) structural re-arrangements such as gene duplications and deletions. These structural re-arrangements lead to copy number variation (CNV), or gene fusions caused by translocation, deletions, or inversions.
2. In contrast, epigenetic modifications (e.g., DNA methylation and histone modification) impact gene expression and/or function without changing the underlying DNA sequence.

Cancer research relies on the ability to find these alterations from both traditional (tissue/cell) as well as liquid biopsy samples. DNA and RNA for next-generation sequencing (NGS) are typically extracted from tissue samples that were removed surgically from solid tumors, either fresh frozen (FF) or Formalin-Fixed, Paraffin-Embedded (FFPE), or from cells obtained by fine needle aspiration. Alternatively, circulating cell-free or tumor nucleic acids (cfDNA or ctDNA) are isolated from blood, plasma, or other bodily fluids. In both instances, DNA samples are extremely precious, available in limited quantities, and are often of poor or variable quality. Methods such as laser-capture microdissection or other forms of tissue dissection (for solid tumor FFPE samples), or fluorescence-activated cell sorting (FACS) of blood cells may be used to enrich for the proportion of cancer cells in a sample, but further reduce the amount of DNA or RNA available for NGS library construction.

As the first step in the NGS workflow, sample preparation holds the key to unlocking the potential of every somatic oncology testing sample. Since these samples are precious and challenging, highly efficient sample preparation solutions are needed to preserve sample integrity and convert nucleic acids into sequenceable molecules with minimal loss and bias. Roche Sample Prep Solutions offer KAPA EvoPrep Boost Kits and KAPA EvoPlus Boost Kits for DNA library construction, and KAPA RNA EvoPrep Kits for the preparation of libraries for RNA sequencing. These kits may contain amplification modules, which include KAPA EvoAmp ReadyMix powered by KAPA HiFi HotStart DNA Polymerase for high-efficiency, low-bias, high-fidelity library amplification. KAPA EvoPrep Kits may also be combined with KAPA HiFi Uracil+ ReadyMix in epigenetic oncology studies that involve methyl-conversion of DNA using various methods.

Our highly optimized library preparation kits and engineered enzymes enable high library complexity, lower duplication rates, and more uniform coverage, which is particularly important in somatic oncology research. The KAPA library preparation kits are ideal for somatic oncology research for the following reasons:

Challenging sample types, including plasma cfDNA, FFPE, urine cfDNA, and cerebrospinal fluid (CSF) cfDNA, yield nucleic acid of variable quality and quantity. Depending on the need for DNA fragmentation, these samples can be processed successfully using library prep reagents like either KAPA EvoPrep Boost Kits or KAPA EvoPlus Boost Kits. Recent oncology research has shown how this can be used to inform cancer disease monitoring when performing both low-pass whole genome sequencing (WGS) as well as targeted sequencing of cfDNA from CSF samples.¹ The robust and flexible chemistry employed by the KAPA library prep kits can be further optimized for challenging sample types by using an overnight ligation step to generate NGS libraries prepared from different samples (plasma DNA and tumor, germline and cell line genomic DNA), with very high success rates.² Further modifications to the protocol, such as shearing conditions and increased adapter molar ratios, allow for high-quality data to be generated from low input amounts.³

Tumors are heterogeneous, consisting of many subpopulations of clonal cells. Mutations of interest may be present at very low frequencies, thus requiring deep sequencing (high coverage) to achieve high-confidence variant calls. In addition to low-frequency variants, specimens with a low percentage of tumor tissue (high levels of normal cell contamination) also require high sensitivity. The highly optimized, integrated workflow using both KAPA library preparation kits and Roche target enrichment solutions provides efficient sample preparation solutions for circulating tumor DNA (ctDNA) librarie.^4,5 Use of the KAPA EvoAmp ReadyMix in combination with the KAPA library prep and target enrichment solutions supports the robust detection of genomic alterations in ctDNA at low allele frequencies while minimizing artifacts.⁶ The KAPA EvoPlus Boost Kit, which includes the KAPA EvoAmp ReadyMix for the amplification of NGS libraries, also addresses the challenges of sequencing samples with limited tumor content or low purity.⁶

While high sensitivity of enzymes is important in somatic oncology research applications, this must be combined with a low error rate in order to ensure accurate variant detection. KAPA HiFi DNA Polymerase—which is the enzyme in the KAPA EvoAmp ReadyMix library amplification kits—has one of the lowest published error rates^7,8 and is well suited for applications that require high-fidelity enzymes. High variant detection rates have been demonstrated with the KAPA EvoPrep Boost Kit with KAPA EvoAmp ReadyMix for library amplification when using variable quality FFPE⁹ and ctDNA¹⁰ material.

Accurate sample quantitation is a key step in generating libraries for sequencing FFPE-derived DNA.¹¹ For low-quality samples, the KAPA NGS FFPE DNA QC Kit enables the successful quality assessment of genomic DNA (gDNA) extracted from FFPE samples.

Roche Sample Prep Solutions provide workflows that enable high sequencing efficiency for somatic oncology research applications. High library complexity, low duplication rates, and uniform coverage (low bias) contribute to lower sequencing costs by sequencing the information that matters. By delivering high library complexity, low duplication rates, and uniform coverage, these optimized protocols reduce overall sequencing costs by sequencing the information that matters.