Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Abstract

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An Integrated Next-Generation Sequencing System for Analyzing DNA Mutations, Gene Fusions, and RNA Expression in Lung Cancer

“…DNA hotspots were prioritized according to the mutation prevalence and level of evidence supporting targetable therapy options in NSCLC. The DNA NGS library covers 55 hotspot regions in 20 genes including EGFR , KRAS , STK11 , PIK3CA , TP53 , and others (Table 2) and is based on previously published methods [25], [26], [27]. Development and validation of the RNA NGS panel are described extensively in previously published work [28].…”

“…TNA samples were stratified based on resulting amplifiable copies per μl. FNA smear TNA isolations followed the targeted DNA-Seq protocol for sample inputs described in previous work [27] to attain a target of 400 amplifiable DNA template copies per reaction. RNA libraries were not prepared from FNA smears as only one sample yielded sufficient RNA for evaluation.…”

“…Unassociated amplicons were filtered prior to a second pass alignment against the GRCh37 reference using bwa-mem v0.6.1 (using the following options: -O 5,5 -E 1,1) [36]; local realignment and QScore recalibration were performed using GATK v1.3-21 [37]. INDEL calling was performed based on the % variant allele frequency (VAF) and coverage depth with empirically determined thresholds [27]. A decision tree algorithm trained on an independent cohort of 400 FFPE samples was used to call SNVs [27].…”

Use of the GeneReader NGS System in a clinical pathology laboratory: a comparative study

“… 1 As such, oncologists are increasingly requesting molecular testing of cancer samples. In addition, in a clinical pathology laboratory, targeted NGS has the advantages of low sample input, 1 2 high volume throughput and the ability to simultaneously interrogate multiple genes. 3 For biopsy-based cancer diagnosis, where the tissue source is often limited, the ability to gain maximum insights from minimal sample material is particularly beneficial.…”

“… 3 For biopsy-based cancer diagnosis, where the tissue source is often limited, the ability to gain maximum insights from minimal sample material is particularly beneficial. 2 As such, many pathology laboratories such as ours are faced with a pressing need to establish an NGS workflow to meet these requirements.…”