Clinical Diagnostics
by Natalie A. LaFranzo
Reference Standards Address Technical Challenges in DNA Resequencing
T
he extensive workflow required to analyze DNA samples using next-generation sequencing (NGS) leaves many opportunities for the introduction of variability. To achieve accurate and precise results in an NGS assay, each stage of the protocol must be routinely optimized, validated and monitored. In this regard, well-characterized reference standards are an effective route to earlier detection of dis- eases, noninvasive monitoring and lower sample input, all of which push the technical limits of NGS.
Challenges of next-generation sequencing NGS has become a universal tool in many industries, and most recently
moved into the clinical arena for use in disease diagnosis. Laboratories need to optimize and validate their workflow in order to analyze patient- derived materials. They must demonstrate that instruments, apparatus, reagents and personnel comply with Clinical Laboratory Improvement Amendments (CLIA) guidelines and oversight by organizations such as the College of American Pathologists (CAP) and the American College of Medical Genetics and Genomics (ACMG) to ensure accuracy and consis- tency. As an example, the minimum data set requirements from the New York State guidelines are shown in Table 1.1
Meeting the evolving needs of NGS Options that satisfy validation and optimization needs include cell lines,
reference standards, patient samples and oligonucleotides. Testing methodologies are recommended based on a laboratory’s accreditation or requirements. New York State guidelines state that researchers must “Establish the analytical sensitivity of the assay for each type of variant detected by the assay. This can initially be established with defined mixtures of cell line DNAs (not plasmids), but needs to be verified with 3–5 patient samples.”1
The NGS workflow challenges are:
• Tumor sample—heterogeneity (stromal contamination), low quantity and poor sample quality are important factors that impact the final assay results
Table 1 – NGS minimum data set requirements from the New York State guidelines for somatic genetic variant detection
• DNA extraction—extraction from low-quality and low-quantity samples and accurate assessment of quantity are challenges presented by patient-derived samples
• Library preparation—specific library preparation approaches are tailored to the goals of the experiment and the quantity and quality of the sample
• Sequencing—read length and type (paired-end vs single-end) and sample multiplexing are determined by the library fragment size and the coverage required for detection
• Bioinformatics—on-sequencer analysis may be employed, or data may be exported into either commercially available software or a laboratory-built pipeline
• Analysis and interpretation—after generating a list of variant calls and their corresponding frequencies, database annotations, statistics and metadata may be incorporated to improve understanding and interpretation of assay results.
Preanalytical variability
Tumor sample and DNA extraction High-quality genetic material is needed to generate interpretable data. Sample age, method of preservation and storage conditions all impact quality. Suitable extraction kits and methods are determined by sample quality. For example, consider formalin-fixed, paraffin-embedded (FFPE) tissue samples. These samples must be extracted using a technique that first removes the paraffin via chemical dissolution and that can accom- modate degraded genomic material. When using diagnostic-grade kits such as the QIAamp DSP DNA FFPE tissue kit (QIAGEN, Valencia, Calif.), certain parameters may need to be optimized before an approved proto- col can be established. During optimization, a well-characterized standard is used to evaluate how protocol modifications affect the resulting DNA yield. One such product, FFPE reference standards from Horizon Discovery (Cambridge, U.K.), are prepared using highly homogeneous cell densities that yield a consistent and reproducible quantity of DNA. (Note: Horizon Discovery reference standards are approved for research use only.) Data from Horizon shows that when DNA is extracted from these standards using five different commercially available protocols, significant variability in yield is observed, as shown in Figure 1. Use of a cell-line-derived, renewable refer- ence standard helps ensure that patient samples are not compromised.
Following extraction, accurate DNA yield must be determined in order to input the appropriate amount of material for downstream sequencing library preparation, as well as to understand theoretical allele frequency thresholds (see below). While spectroscopic methods like UV/VIS provide adequate estimation at concentrations higher than 10 ng/µL, fluorometric
AMERICAN LABORATORY • 14 • AUGUST 2015
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