AUTOMATED TARGETED SEQUENCING WORKFLOW
End-to-End, Automated Targeted Sequencing Workflow
The NEXTFLEX® NGS Hybridization Panels are part of a comprehensive workflow for genetic analysis offered by PerkinElmer, a single-source solution that includes nucleic acid isolation, NGS library preparation, QC, data analysis, and data interpretation. Designed to simplify targeted NGS, PerkinElmer’s end-to-end workflow delivers accurate and reliable results with the flexibility to meet variable throughput. Automated and manual workflow modules are available to meet the needs of the lab. Traditional sequencing-based testing uses invasive venipuncture to collect whole blood, but advancements in PerkinElmer’s DNA extraction technology enables enough high-quality extraction from a dried blood spot (DBS) for targeted sequencing. Thus, a single DBS can be used for both enzymatic and genetic testing without the need of resampling.
NEXTFLEX® NGS HYBRIDIZATION PANEL WORKFLOW
SAMPLE COLLECTION
DNA EXTRACTION
DNA QC
LIBRARY PREP AUTOMATION
LIBRARY QC
SEQUENCING
RESULT ANALYSIS
SAMPLE COLLECTION
DNA EXTRACTION
DNA/LIBRARY QC
TARGET CAPTURE
LIBRARY PREP AUTOMATION
NEXTFLEX® Hybridization Panels
The NEXTFLEX® DMD NGS Hybridization Panel identifies relevant mutations in the dystrophin gene (Table 1A), the NEXTFLEX® LSD NGS Hybridization Panel identifies mutations present in the exons of 11 genes associated with LSDs (Table 1B), and the NEXTFLEX® Core Exome NGS Hybridization Panel identifies mutations present in ~6,000 genes associated with common genetic disorders (Table 1C). The targeted region for each gene includes the exons with at least five base pair flanking.
Table 1. Genes included in the NEXTFLEX® NGS Hybridization Panels. NEXTFLEX® CoreExome NGS Hybridization Panel
| Targets | Disease |
|---|---|
| DMD | Duchenne muscular dystrophy |
| ARSB | Maroteaux-Lamy syndrome |
| GAA | Pompe disease |
| GALC | Krabbe disease |
| GALNS | Morquio syndrome type A |
| GLA | Fabry disease |
| GLB1 | Morquio syndrome type B |
| GUSB | Mucopolysaccharidosis type VII |
| IDS | Hunter syndrome |
| IDUA | Hurler syndrome |
| SMPD1 | Niemann-Pick disease, type A and B |
| SUMF1 | Multiple sulfatase deficiency |
| ~6,000 genes | Common genetic disorders |
Verification Study
Study Samples
A variety of samples (Table 2) were processed using the DMD, LSD, and Core Exome panels and sequenced on three separate days.
Table 2. Samples used in the verification study.
| RUN | Sample # | Sample ID | Sample Type | Library to Use | Mutation Status |
|---|---|---|---|---|---|
| 1 | 1 | Sample 1 | DBS | Core Exome | Population Normal |
| 1 | 2 | NA12878 | Culture | Core Exome | HapMap Sample |
| 1 | 3 | NA12891 | Culture | Core Exome | HapMap Sample |
| 1 | 4 | Sample 1 | DBS | DMD | Population Normal |
| 1 | 5 | NA12878 | Culture | DMD | HapMap Sample |
| 1 | 6 | NA23099 | Culture | DMD | Female DMD EX8-17DUP |
| 1 | 7 | Sample 1 | DBS | LSD | Population Normal |
| 1 | 8 | NA040372 | Culture | LSD | GALC: 30KB deletion |
| 1 | 9 | NA12878 | Culture | LSD | HapMap Sample |
| 2 | 1 | Sample 1 | DBS | Core Exome | Population Normal |
| 2 | 2 | NA12878 | Culture | Core Exome | HapMap Sample |
| 2 | 3 | NA12891 | Culture | Core Exome | HapMap Sample |
| 2 | 4 | Sample 1 | DBS | DMD | Population Normal |
| 2 | 5 | NA03929 | Culture | DMD | Male DMD EX46-50DEL |
| 2 | 6 | NA23159 | Culture | DMD | Male DMD EX17DUP |
| 2 | 7 | Sample 1 | DBS | LSD | Population Normal |
| 2 | 8 | NA04372 | Culture | LSD | GALC: 30KB deletion |
| 2 | 9 | NA13878 | Culture | LSD | HapMap Sample |
| 3 | 1 | Sample 1 | DBS | Core Exome | Population Normal |
| 3 | 2 | NA2878 | Culture | Core Exome | HapMap Sample |
| 3 | 3 | NA12892 | Culture | Core Exome | HapMap Sample |
| 3 | 4 | Sample 1 | DBS | DMD | Population Normal |
| 3 | 5 | NA12878 | Culture | DMD | HapMap Sample |
| 3 | 6 | Sample 2 | Culture | DMD | Female DMD del 49-50 |
| 3 | 7 | Sample 1 | DBS | LSD | Population Normal |
| 3 | 8 | NA04372 | Culture | LSD | GALC: 30 KB deletion |
| 3 | 9 | NA12878 | Culture | LSD | HapMap Sample |
Performance Verification
DNA isolated from DBS and cell culture samples were used for comparison between sample types and verification against data generated from a workflow previously validated by PerkinElmer Genomics Laboratory.
Analysis of NGS sequencing data involved BWA alignment of reads to the human genome (hg37/19). Panel-specific BED files were used to bin reads as “on” or “off” target based on genomic locations of targeted reads. Depth of coverage was calculated from aligned reads on a per base basis. Variant calling was accomplished using SnpEff and SnpSift open-source bioinformatics tools. CNV analysis was accomplished by visualizing read depth across gene target regions.
Materials and Methods
The NEXTFLEX® NGS Hybridization Panels contain library prep reagents, index barcodes, blockers, baits, streptavidin-beads, hybridization and wash buffers, and post-capture amplification reagents. The kit is designed for approximately 2.5-hour precapture DNA library construction, with enzymatic fragmentation included, from as little as 1 ng DNA, and is optimized for subsequent target enrichment prior to sequencing. The kit can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina® platforms. The recommended minimum input for hybrid capture is 100 ng of starting material into the pre-capture library prep.
Results
Read Count
The global sequencing metrics of the NEXTFLEX® NGS Hybridization Panels were evaluated using three unique samples analyzed in three separate runs. The number of total reads (Figure 2A) and other global sequencing metrics were observed to be similar in cell culture samples and DBS samples.
Unique vs Duplicate reads
The average duplicate read rate was approximately 12% (Figure 2B). Duplicates are unavoidable when sequencing PCR-amplified fragmented DNA, but a low percentage of duplicate reads results in more usable sequencing data. The low percentage of duplicate reads also enables more accurate measurement of CNVs. CNVs are detected visually in DMD (Figure 4, 5), LSD (Figure 6), and Core Exome (Figure 7) target regions.
Percentage of Reads Mapping On-Target
The specificity of targeted NGS assays is demonstrated by the high percentage of on-target reads. In this study, the percentage of on-target reads was more than 60% (Figure 2C). Depth of coverage for on-target reads is ~100X or greater for all three NGS Hybridization Panels (Figure 2D). All three panels achieved significantly greater than 20X coverage across the targeted regions of interest. Coverage statistics can be visualized more comprehensively using the NEXTFLEX® Data Analysis Platform (Figure 3).
Figure 2. (A) Total reads, (B) duplicate read rate, (C) on-target reads and (D) average read coverage in cell culture and DBS samples with NEXTFLEX® NGS Hybridization Panels
Figure 3. Screen-capture depicts a visualization of coverage statistics for a sample processed with the NEXTFLEX® Lysosomal Storage Disorders NGS Hybridization Panel using the NEXTFLEX® Data Analysis Platform. Line graphs shows percent of bases fully covered at different minimum coverage thresholds.
Figure 4. CNV Visualization of the DMD gene. A, B, C. Visualization of a normal sample processed through three separate sequencing runs. D. Visualization of a heterozygous deletion spanning exons 49 and 50 in a female sample. E. Visualization of a heterozygous duplication spanning exons 8 through 17 in a female sample. F. Visualization of a deletion spanning exons 46 through 50 in a male sample. CNV events are highlighted with superimposed red boxes.
Figure 5. Screen-capture depicts a visualization of coverage for The DMD sample, NA03929, using the NEXTFLEX® Data Analysis Platform in the left pane and SNVs detected in the right pane.
Figure 5. CNV visualization of the GALC gene of the LSD Panel. A, B, C. Visualization of a normal sample processed through three separate sequencing runs. D and E. Visualization of a heterozygous 30 kb deletion in the GALC gene highlighted in red. The relative coverage halves in value for the exons compromised by the 30 kb deletion and indicates a single copy for the region.
Figure 7. CNV visualization of a gene in the NEXTFLEX® NGS Core Exome Panel using the NEXTFLEX® Data Analysis Platform. Dots near red line indicate CNV heterozygous deletion.
Comparison to a Validated PerkinElmer Genomics Workflow
The NEXTFLEX® NGS Hybridization Panel workflows were compared to the previously validated PerkinElmer Genomics workflows by using both methods to detect the polymorphisms in the verification study sample set. A significant overlap in the polymorphisms detected with these two methods was observed, indicating a high concordance between the NEXTFLEX® NGS Hybridization panel workflows and the previously validated PerkinElmer Genomic workflows. Core Exome workflow data was used as a model for polymorphism comparison because the workflow had the largest set of targets (Figure 8). Additionally, this comparison uses Genome in a Bottle related samples, which allows for an analysis to only identify expected polymorphisms.
Figure 8. A, B. Venn diagram visualizations of NEXTFLEX® Core Exome NGS Hybridization panel compared with the previously validated PerkinElmer Genomics core exome panel, as assessed by their ability to detect polymorphisms. A and B represent the same NA12891 libraries sequenced on separate sequencing runs.
Summary
PerkinElmer® NEXTFLEX® NGS Hybridization Panels (RUO) are reliable tools for the detection of CNVs and SNPs in genes associated with Duchenne Muscular Dystrophy, Lysosomal Storage Disorders, and other common genetic disorders. Performance of the NEXTFLEX®NGS Hybridization Panel workflows were verified against a previous validated workflow to detect CNVs and SNPs reproducibly.
References
- Platt, FM. Emptying the stores: lysosomal diseases and therapeutic strategies. Nat Rev Drug Discov. 2018;17(2):133-150. (doi: 10.1038/nrd.2017.214)
- Schielen, PCJI, Kemper EA, Gelb, MH. Newborn Screening for Lysosomal Storage Diseases: A Concise Review of the Literature on Screening Methods, Therapeutic Possibilities and Regional Programs. Int J Neonatal Screen. 2017;3(2). (doi: 10.3390/ijns3020006)