NGS Long-Range PCR Large Deletion Detection

NGS Long-Range PCR Large Deletion Detection

1. Technical Background

With the widespread clinical application of CRISPR and other gene-editing technologies, their potential safety risks have drawn increasing attention from regulatory agencies worldwide. DNA double-strand breaks (DSBs) generated by CRISPR cleavage can lead to chromosomal rearrangement events during the repair process, including large chromosomal deletions, insertion of exogenous DNA fragments, and chromosomal translocations. The U.S. Food and Drug Administration (FDA) has explicitly emphasized the importance of chromosomal rearrangement detection, as these events may cause serious adverse outcomes including genomic instability, elevated disease risk, treatment failure, and other unpredictable consequences.

Figure 1. FDA guidance document Human Gene Therapy Products Incorporating Human Genome Editing: key recommendations on the detection of chromosomal structural abnormalities

The NGS-based Long-range PCR chromosomal rearrangement detection platform developed by ZhuHai GeneRulor is specifically designed for comprehensive detection of multiple structural variant events induced by gene editing, including large deletions (≥50 bp), small deletions (<50 bp), and insertions of various sizes. The technology employs site-specific primers designed approximately 5 kb upstream and downstream of the cut site to amplify long DNA fragments spanning the cleavage locus (approximately 10 kb), combined with NGS technology to enable comprehensive capture and precise quantification of all editing events. As a complementary approach to PEM-seq, NGS-based Long-range PCR addresses the limitations of existing methods in large deletion detection, providing comprehensive and reliable data support for the safety evaluation of gene therapy products.

2. Detection Principle of NGS-Based Long-Range PCR

NGS-based Long-range PCR is a highly sensitive detection method specifically designed for large deletion events following gene editing. Its core principle involves designing site-specific primers approximately 5 kb upstream and downstream of the target cut site to amplify long DNA fragments spanning the cleavage locus (approximately 10 kb), thereby effectively capturing various gene-editing products including large deletions (≥50 bp), small deletions (<50 bp), and insertions of all sizes. The detailed detection workflow comprises the following steps:

1. Design site-specific primers approximately 5 kb flanking the cut site to cover a genomic region of ~10 kb;

2. Use a high-fidelity long-range PCR polymerase to specifically amplify the post-editing DNA samples;

3. Subject purified long-range PCR products to ultrasonic random fragmentation;

4. Construct a standard sequencing library from the fragmented DNA;

5. Perform deep sequencing of the library by high-throughput sequencing technology;

6. Apply a professional bioinformatics analysis pipeline to precisely detect and quantify all structural variant events.

Figure 2. Experimental workflow of NGS-based Long-range PCR

3. Detection Advantages of NGS-Based Long-Range PCR

3.1 Comprehensive Capture of Multiple Gene-Editing Events

Compared with other technologies, NGS-based Long-range PCR demonstrates significant advantages in structural variant detection:

1. Broad detection coverage: simultaneous detection of large deletions (≥50 bp), small deletions (<50 bp), and insertions of all sizes;

2. High capture efficiency: direct amplification of the target region using site-specific primers, improving the capture efficiency of all categories of structural variant events;

3. Accurate quantification: combined with high-throughput sequencing technology to achieve precise quantification of all deletion and insertion events;

4. Differential amplification advantage: during PCR amplification, DNA fragments carrying large deletions are relatively shorter and are amplified with higher efficiency, facilitating the detection of low-frequency deletion events; the method also demonstrates good capture capability for insertion events.

3.2 Technical Complementarity

The FDA has explicitly noted that PEM-seq technology may fail to effectively detect large deletion events beyond the primer design region (50–110 bp from the cut site) due to the proximity of primer design to the cleavage locus, resulting in a detection blind spot. NGS-based Long-range PCR addresses this technical limitation by designing primers at a distal position (~5 kb from the cut site), enabling comprehensive detection of all categories of deletion and insertion events. Together with PEM-seq, these two technologies form a complementary pair that collectively build a complete gene-editing safety evaluation framework.

3.3 Service Advantages

1. Comprehensive structural variant analysis: simultaneous full-panel detection and analysis of large deletions, small deletions, and insertion events; specialized primer design providing individualized primer solutions for different editing sites to ensure amplification specificity and genomic coverage;

2. Standardized workflow: from sample processing to library construction, the entire process follows a standardized workflow to ensure result reproducibility;

3. Deep sequencing: high-throughput sequencing platform enables deep sequencing, improving the detection sensitivity for low-frequency variants;

4. Specialized data analysis: application of professional gene-editing software tools such as CRISPR-LargeDel for data evaluation and analysis, with false-positive filtering to deliver precise editing efficiency quantification;

5. Comprehensive report delivery: provision of a full analytical report covering statistics for all categories of deletion and insertion events, functional annotation, and more;

6. Regulatory compliance assurance: meets technical requirements of FDA and other regulatory agencies for gene-editing safety evaluation; supports IND submission.

4. Technical Application Scenarios

4.1 Application Fields

1. Comprehensive safety evaluation:   Quantification of all structural variant events (deletions and insertions) induced by gene editing to assess potential safety risks;

2. Target site screening optimization:   Comparison of structural variant patterns and risks across different target sites or sgRNA designs to identify the optimal editing strategy;

3. Editing efficiency assessment:   Precise quantification of the proportions of different editing event types for a comprehensive evaluation of gene-editing efficiency;

4. IND submission data support:   Provision of gene-editing safety data compliant with regulatory requirements, supporting drug development at all stages;

5. Quality control:   Serving as a key quality indicator for gene therapy products to ensure product safety.

4.2 Complementary Application with PEM-seq Technology

NGS-based Long-range PCR and PEM-seq are mutually complementary technologies that together form a comprehensive gene-editing safety evaluation framework:

1. PEM-seq: focused on chromosomal translocation event detection, identifying editing-induced chromosomal rearrangements, translocations, and off-target cut sites;

2. NGS-based Long-range PCR: specialized for detection of all categories of deletions (large deletions ≥50 bp and small deletions <50 bp) and insertion events, addressing the limitations of PEM-seq in structural variant detection;

3. ZhuHai GeneRulor recommends the concurrent use of both technologies in gene-editing safety evaluations to obtain the most comprehensive dataset of genomic structural variants.

4.3 Representative Success Cases

ZhuHai GeneRulor has continuously provided comprehensive gene-editing safety evaluation services — encompassing NGS-based Long-range PCR detection, data analysis, and regulatory document preparation — to companies including Iaso Bio and Shansha Bio, supporting the successful FDA IND submissions and regulatory approval of their CAR-T products. In addition, we have provided gene-editing safety evaluation services to numerous domestic and international gene therapy companies, supporting IND submissions and clinical translation of multiple products.

5. NGS-Based Long-Range PCR Detection Report Content

The NGS-based Long-range PCR analytical reports provided by ZhuHai GeneRulor adopt a standardized format to ensure professional and comprehensive data presentation. The introductory section covers project background, NGS-based Long-range PCR technical principles, library construction workflow description, and bioinformatics analysis pipeline overview, providing clients with the necessary technical context. This is followed by sample information and sequencing data statistics, which include sample metadata (sample name, experimental/control group classification, sgRNA sequences, genomic coordinates, and primer sequence information), raw sequencing data quality metrics, and comparative reference genome alignment results, ensuring data quality and reliability. In addition, the report includes the following key components:

(1) Large Deletion (Large Deletion) Event Statistics for Experimental and Control Groups:   Comprehensive statistics of large deletion events (≥50 bp) in both the experimental and control groups, displaying the distribution of deletion events across different size ranges (50–100 bp, 100–200 bp, 200–1,000 bp, ≥1,000 bp), and providing the large deletion status for each experimental sample after background subtraction through control group filtering.

Figure 3. NGS-based Long-range PCR representative report: Large Deletion event statistics for experimental and control groups

Figure 4. NGS-based Long-range PCR representative report: Large Deletion events in experimental samples after false-positive filtering

(2) Large Deletion Size Distribution Visualization:   Deletion size distribution plots and symmetry distribution plots to visually display the relative positional relationship between deletion events and the cut site.

Figure 5. NGS-based Long-range PCR representative report: symmetry distribution plot of large deletion sizes

Figure 6. NGS-based Long-range PCR representative report: symmetry distribution plot of large deletion positions

(3) Large Deletion Event Characterization and Annotation:   Detailed characterization and functional annotation of large deletion events, including chromosomal position, deletion size, distance from the cut site, and the gene regions involved.

Figure 7. NGS-based Long-range PCR representative report: large deletion event characterization and annotation

(4) Large Deletion Event Impact Annotation:   In-depth functional impact assessment of large deletion events, including annotation of variant types such as transcript_ablation and frameshift_variant, classified by impact severity level (HIGH, MODERATE, LOW), with particular attention to the potential effects on functionally important genes such as tumor suppressor genes (TSGs)

Figure 8. NGS-based Long-range PCR representative report: large deletion event impact annotation

(5) Small Indel Event Analysis:   Precise statistics of small deletion (<50 bp) and insertion events of various sizes in both experimental and control groups. Small deletions are further subdivided into 1–20 bp and 20–50 bp size ranges; insertions are subdivided into 1–10 bp, 10–20 bp, and ≥20 bp size ranges. This provides a comprehensive display of the pattern and frequency of small indel editing events. Through background subtraction of events detected in the control group, the true small indel frequency in experimental samples is precisely calculated, providing reliable data support for evaluating gene-editing precision and efficiency.

Figure 9. NGS-based Long-range PCR representative report: small indel event analysis before and after filtering

6. NGS-Based Long-Range PCR Service Content

* Service turnaround: standard workflow 20–30 business days.

* Service highlight: integrated end-to-end service support from sgRNA design through final data analysis is available.

7. Sample Requirements

* Notes: (1) All samples must meet the above standards to ensure the accuracy and reliability of detection results. (2) Clients may also submit cells for DNA extraction; cell quantity requirements: >1×10⁶ cells per site. (3) For special sample types, please contact the ZhuHai GeneRulor technical team in advance (Tel: 400-6309596; Order/Technical Support: service@generulor.com).

8. References

[1] Jia H, Guo Y, Zhao W, Wang K. Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer. Sci Rep. 2014;4:5737.

[2] Keith. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2021;523:481–485.

[3] Tsai SQ, Zheng Z, Nguyen NT, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33(2):187–197.

[4] Jia H, Guo Y, Zhao W, Wang K. Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer. Sci Rep. 2014;4:5737.

[5] Hwang GH, Lee SH, Oh M, Kim S, Habib O, Jang HK, ... & Bae S. (2024). Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases. Nature Biomedical Engineering.

[6] Kosicki M, Tomberg K & Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765–771 (2018).