This article covers analytical principles of cancer next generation sequencing (NGS). Cancer samples require special considerations due to the cancer-specific applications of testing, as well as cancer sample specific issues, including low input, low tumor purity, or fixation-related artifacts. Laboratories typically use a combination of approaches around specimen processing, assay design, and bioinformatics analysis to allow for successful detection of actionable biomarkers. Examples of these approaches for cancer NGS testing are discussed and reviewed here.
Molecular profiling of tumor samples by next generation sequencing may be challenging as tumor samples may be small or compromised due to storage and fixation issues, and frequently have admixed normal.
During the assay design process, steps may be taken during sample processing, library preparation, and bioinformatics analysis steps to address the unique challenges and clinical questions for somatic molecular testing.
Control samples and quality control metrics should be monitored to assess the performance of the assay to detect somatic variants.
Molecular profiling and biomarker detection in tumor samples by next generation sequencing (NGS) provide key information that may be used to guide patient management and therapy selection that allows the delivery of personalized medicine. NGS or “massively parallel” sequencing are the terms used to refer collectively to a range of sequencing technologies that involve analysis of large numbers of different DNA sequences in parallel (ie, in a single reaction). The past decade has seen an exponential increase in novel methodologies in template generation and sequencing that were developed to address a range of questions in clinical practice and cancer research. As an example, tumor mutation burden (TMB), defined as the number of mutations per megabase of sequence, predicts response to immune checkpoint inhibitor therapy. TMB is readily detectable using large NGS-based cancer panels or cancer whole exome sequencing (WES) studies. Other biomarkers detectable by NGS include point mutations, insertion/deletion mutations, copy number alterations, and gene fusions. These biomarkers can support the diagnosis of malignancy, inform prognosis, and identify therapeutic targets.
Optimization and validation of the performance characteristics of a cancer NGS assay is critical for the accurate detection of biomarkers. The analytical phase of NGS testing typically encompasses steps including DNA fragmentation, end repair, adapter ligation, library enrichment, sequencing, and bioinformatics analysis. DNA fragmentation is, generally, done by mechanical shearing (eg, sonication or nebulization) or enzymatic digestion. This step produces fragments of a size compatible with short read sequencing lengths. The end repair step is required following fragmentation if the fragmentation method does not produce blunt ends. The end repair step fills in 5′ overhangs, trims 3′ overhangs, and phosphorylates the 5′ nucleotide to provide a blunt end to which adapters can ligate. The end repair step is generally not required if polymerase chain reaction (PCR) amplification is used for library enrichment of target sequences. As part of library preparation, the enrichment step can enhance the coverage of specific genetic regions such as cancer-specific genes or all coding exons in the case of WES. For whole genome sequencing (WGS), the complete genomic DNA is sequenced and hence no enrichment step is required. Following preparation of libraries and addition of patient-specific barcodes, the libraries are pooled and loaded onto the sequencer. Sequence data is bioinformatically processed with steps including demultiplexing to assign reads to patient samples, alignment to a reference genome, variant calling, and annotation.
There are specific considerations for the analysis of tumor samples, due to the nature of tumor samples, the frequent presence of admixed normal, and the clinical context and clinical utilization of the assay at each step in the analytical phase of cancer NGS testing ( Fig 1 ). The analytical principles for cancer NGS will be the main focus of this article.