Somatic oncology research focuses on the study of cancers resulting from DNA alteration that occurred after conception; i.e., alteration that was not inherited. Two types of DNA alterations exist:
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 (and then frozen, or fixed in formalin and embedded in paraffin; FFPE), or 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 reduces 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 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 HyperPrep Kits and KAPA HyperPlus Kits for DNA library construction, and KAPA RNA HyperPrep Kits for the preparation of libraries for RNA-Seq. These kits include KAPA HiFi HotStart DNA Polymerase for high-efficiency, low-bias, high-fidelity library amplification. KAPA HyperPrep Kits may also be combined with KAPA HiFi Uracil+ ReadyMix in epigenetic oncology studies that involve bisulfite treatment of DNA.
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. The KAPA library preparation kits are ideal for somatic oncology research for the following reasons:
Robust and flexible chemistry
Challenging sample types, including cfDNA, FFPE and cerebrospinal fluid (CSF) yield DNA of variable quality and quantity. Depending on the need for DNA fragmentation, these samples can be processed successfully using either KAPA HyperPrep Kits or KAPA HyperPlus Kits, as shown in the cancer panel sequencing of tumor genome 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
Highly sensitive detection
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) libraries.4,5 Use of KAPA HiFi enzyme allows for sensitive detection of genomic alterations in ctDNA at low allele frequencies, while minimizing false positives calls.6 KAPA HyperPlus Kit, which includes KAPA HiFi DNA Polymerase for the amplification of NGS libraries, also addresses the challenges of sequencing samples with limited tumor content or low purity.7
Low error rate
While high sensitivity of enzymes is important in somatic oncology applications, this must be combined with low error rate in order to ensure accurate variant detection. KAPA HiFi DNA Polymerase has one of the lowest published error rates8,9 and is well suited for applications that require high fidelity enzymes. High variant detection rates have been demonstrated with the KAPA HyperPrep Kit with KAPA HiFi HotStart DNA Polymerase for library amplification when using variable quality FFPE10 and ctDNA11 material.
Accurate sample quantitation is a key step in generating libraries for sequencing FFPE-derived DNA.12 For low quality samples, the KAPA hgDNA Quantification and QC Kit qPCR assay enable the successful quantitation and quality assessment of genomic DNA (gDNA) extracted from FFPE samples.
Roche Sample Prep Solutions provide workflows that enable high sequencing efficiency for somatic oncology applications. High library complexity, low duplication rates and uniform coverage (low bias) contribute to lower sequencing costs by sequencing the information that matters. With optimized sample preparation workflows, NGS sequencing has the potential to guide personalized cancer therapy, monitor residual disease, and track the evolving tumor genome.
Pan, W. et al. (2015) ‘Brain Tumor Mutations Detected in Cerebral Spinal Fluid’, Clinical Chemistry, 61(3), pp. 1–10. doi: 10.1373/clinchem.2014.235457.
Newman, A. M. et al. (2014) ‘An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage’, Nature Medicine, (April).
Reichel, J. et al. (2015) ‘Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells’, Blood, 125(7), pp. 1061–1073. doi: 10.1182/blood-2014-11-610436.
Li, H. et al. (2018) ‘Plasma PIK3CA ctDNA specific mutation detected by next generation sequencing is associated with clinical outcome in advanced breast cancer’, American Journal of Cancer Research, 8(9), pp. 1873–1886.
Xu, R. et al. (2018) ‘Sequencing of circulating tumor DNA for dynamic monitoring of gene mutations in advanced non-small cell lung cancer’, Oncology Letters, 15(3), pp. 3726–3734. doi: 10.3892/ol.2018.7808.
Clark, T. A. et al. (2018) ‘Analytical Validation of a Hybrid Capture–Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA’, Journal of Molecular Diagnostics. American Society for Investigative Pathology and the Association for Molecular Pathology, 20(5), pp. 686–702. doi: 10.1016/j.jmoldx.2018.05.004.
Eifert, C. et al. (2017) ‘Clinical application of a cancer genomic profiling assay to guide precision medicine decisions’, Personalized Medicine. Future Medicine Ltd London, UK, 14(4), pp. 309–325. doi: 10.2217/pme-2017-0011.
Quail, MA. et al. (2012) ‘Optimal enzymes for amplifying sequence libraries’, Nature Methods, 9(Dec), pp. 10–11.
Oyola, S. O. et al. (2012) ‘Optimizing Illumina Next-Generation Sequencing library preparation for extremely AT-biased genomes.’, BMC genomics, 13(1), p. 1. doi: 10.1186/1471-2164-13-1.
Lu, H. et al. (2018) ‘Targeted next generation sequencing identified clinically actionable mutations in patients with esophageal sarcomatoid carcinoma’, BMC Cancer. BMC Cancer, 18(1), pp. 1–7. doi: 10.1186/s12885-018-4159-2.
Shu, Y. et al. (2017) ‘Circulating Tumor DNA Mutation Profiling by Targeted Next Generation Sequencing Provides Guidance for Personalized Treatments in Multiple Cancer Types’, Scientific Reports. Springer US, 7(1), pp. 1–11. doi: 10.1038/s41598-017-00520-1.
Marosy, B. A. et al. (2017) ‘Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing’, Current Protocols in Human Genetics. Hoboken, NJ, USA: John Wiley & Sons, Inc., 92(1), p. 18.10.1----18.10.25. doi: 10.1002/cphg.27.