Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing

Summary

This protocol describes an automated, ISO15189-accredited next-generation sequencing workflow for detecting targetable genomic alterations in non-small cell lung cancer (NSCLC) formalin-fixed paraffin-embedded tissues.

Abstract

The success of targeted therapy in non-small cell lung cancer (NSCLC) hinges on the precise identification of driver alterations, including mutations, gene fusions, and amplifications. Next-generation sequencing (NGS) has emerged as a comprehensive molecular diagnostic tool, capable of detecting both known and novel genomic aberrations, providing critical support for personalized NSCLC treatment. However, NGS remains a complex and technically challenging method. Despite its widespread adoption, NGS still faces some challenges, including technical complexity and prolonged turnaround times. Here, the ISO15189-certified NGS workflow implemented in the clinical laboratory is introduced. The standardized protocol encompassed tumor cellularity assessment (≥20 %), DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues (DNA input ≥ 50 ng), automated library preparation, and bioinformatics analysis. By integrating stringent quality control (QC) measures at each step, the workflow ensures high data reliability. Besides, the key innovation in workflow was the automation of NGS library construction. The automated system of NGS library construction included end repair, A-tailing, adapter ligation, hybridization capture, and purification, effectively minimizing human error, enhancing experimental reproducibility, reducing hands-on time, and thus improving efficiency. Together, experience demonstrates that rigorous QC and automated library preparation are essential for maintaining accuracy and scalability in clinical NGS testing. This optimized approach not only ensures compliance with ISO15189 standards but also supports the growing demand for precision oncology in NSCLC management.

Introduction

Non-small cell lung cancer (NSCLC) accounts for 75%-85% of all lung cancer cases, representing both a clinical priority and a therapeutic challenge^1,2,3. In recent years, with the successful development and clinical application of molecularly targeted drugs such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the treatment of advanced NSCLC has entered the era of personalized precision therapies^4,5,6. Targeted therapy offers precise efficacy, high specificity, and minimal side effects^7,8. This shift in treatment has simultaneously increased the demand for molecular diagnostic technologies.

Currently, comprehensive tumor-targeted testing has become an indispensable component of clinical practice that can help elucidate accurate genetic mutation profiles and thus provides a reliable basis for subsequent targeted drug use, drug resistance monitoring, and prognosis assessment for patients. Particularly, NGS-based multi-gene testing, leveraging high-throughput and high-sensitivity technical advantages, has already been recommended for clinical application^9,10,11,12.

Compared to traditional molecular diagnostic techniques, NGS demonstrates groundbreaking advantages. The foremost advantage of NGS lies in its high-throughput capability. It enables the concurrent detection of multiple genes, including both known and unknown genetic alterations^13,14. Meanwhile, NGS achieves comprehensive testing in a single run, relatively reducing cost, sample consumption, and testing turnaround time^15,16. Given these advantages, NGS has been increasingly applied in clinical practice. However, the processes of NGS are complex, including tumor cellularity assessment, nucleic acid extraction, automated library preparation, and bioinformatics analysis. The quality of initial sample processing directly affects the performance of subsequent bioinformatics analyses¹⁷. Especially, library preparation involves a series of steps to convert raw nucleic acid samples into standardized libraries compatible with sequencing instruments. Library preparation includes fragmentation, end repair, adapter ligation, and PCR amplification¹⁸. This complex process often introduces various technical biases: for example, fragmentation may lead to the underrepresentation of heterochromatin regions; the substrate preference of ligase during adapter ligation can result in the loss of certain sequences; and PCR amplification tends to cause uneven coverage in regions with extreme GC content¹⁹. These systemic biases not only reduce the quality of sequencing data but may also lead to erroneous conclusions in subsequent bioinformatics analyses. Therefore, establishing a rigorous QC system is essential to ensure the reliability and consistency of experimental results.

To address these long-standing technical challenges, the laboratory has adopted a fully automated NGS system. The system is designed to reduce manual processing time and decrease overall turnaround periods. It incorporates programmable protocols to ensure both operational flexibility and processing precision while utilizing integrated modules and units to effectively minimize potential technical biases at each step. Furthermore, this automated solution not only improves the reproducibility and throughput of library preparation but, more importantly, ensures that sequencing data more accurately reflects the molecular composition of the original samples. Here, the laboratory would like to introduce the system through the detection of driver alterations using DNA-based NGS.

Protocol

The protocol received an IRB waiver for anonymized archival samples.

1. Pre-testing steps and QC

1. Register sample information in the laboratory information management system (Table 1). Fill out the informed consent form for gene mutation testing.
2. Receive NSCLC FFPE sample. Ensure that the sample used for testing is a paraffin tissue sample that has been stored for no more than 2 years. Ensure that the samples are processed and stored according to standard pathological methods.
   NOTE: This study primarily focuses on the methodological workflow, rather than conducting a large-scale clinical analysis. Therefore, no specific sample number requirement was set for this phase of the research.
3. Cut sections using disposable microtome blades. Change blades, forceps, and 1.5 ml microtubes between samples to prevent cross-contamination and avoid introducing operator-derived contaminants (e.g., hair, skin cells, saliva).
   NOTE: Large sample size (≥ 0.5 cm x 0.5 cm) 6-10 µL, five sheets; small sample size (< 0.5 cm x 0.5 cm) and puncture samples, 6-10 µL, 10 sheets.
4. Perform routine hematoxylin-eosin (HE) staining to evaluate tumor cell content.
5. Select samples containing ≥20% malignant tumor cells for downstream analysis. The tumor content is evaluated by pathologists on HE stained glass slides²⁰.
   NOTE: If the tumor content is less than 20%, under the guidance of the marked tumor regions on the HE slide, scrape off the corresponding tumor tissue area. This process achieves enrichment of tumor tissue by effectively removing surrounding non-neoplastic components, thereby significantly increasing the percentage of tumor cells in the collected material.
6. Perform nucleic acid extraction using an automated purification system as described below.
   1. Section the FFPE block using a microtome to obtain a ribbon. Place the ribbon into a 1.5 ml microcentrifuge tube. Transfer the ribbon to the designated sample input location on the automated extraction instrument.
   2. Use an automatic extraction instrument with an automatic extraction kit and replace manual pipetting with an automatic magnetic bead nucleic acid extraction system.
      NOTE: Automating this step helps ensure automatic extraction to ensure high-precision and reproducible DNA production for downstream NGS applications.
   3. Refer to the instructions of the fully automatic nucleic acid purifier and nucleic acid extraction reagent to extract nucleic acid.
   4. Then, select the C1102 Program, choose sample dewaxing incubation time as 16 h, and elution volume as 100 µL.
      NOTE: If the extracted DNA solution is not immediately tested, it must be stored at -20 °C for no more than 6 months.
7. Assess nucleic acid quality (Table 2) as described below.
   1. Use agarose gel electrophoresis to determine DNA fragment size. Use Agilent2100 and other microfluidic capillary electrophoresis analysis systems for low-input samples.
   2. Dissolve 1 g agarose in 100 mL of TAE solution. Heat the mixture in a water bath until transparent. Cool the solution to around 60 °C.
   3. Connect to the positive electrode (red) on the electrophoresis instrument. Perform negative pole (black) to voltage 180 V, run for 15 min^21,22.
   4. According to the results of agarose gel electrophoresis, categorize the fragment sizes into four levels (A-D; Table 3), and select the nucleic acid of levels A-C for subsequent operations.
   5. Use spectrophotometry to verify purity. Ensure a OD260/OD280 > 1.8; OD260/OD230 > 2.0 to indicate acceptable contaminant levels^23,24.
   6. Use detection kit (Table of Materials) based on sequence-specific fluorescent dyes for fluorometric assay to precisely measure DNA concentration.
   7. Add 1 µL of DNA to be tested to 199 µL of working solution, then mix, briefly centrifuge, and measure its concentration using a fluorometer (Table of Materials).

2. Library preparation and QC (Figure 1)

1. Prepare fragment genomic DNA using ultrasonication (Table of Materials) as described below.
   1. Mix 200 ng genomic DNA in 50 µL of low Tris-EDTA (TE) buffer. Then, keep the sample tube in a pre-cooled (8 °C) sample rack.
   2. Set instrument parameter according to Table 4. Then initiate fragmentation.
   3. Use agarose gel electrophoresis to verify the fragmentation effect. This step generates DNA fragments with damaged ends that require end repair to transform them into blunt ends for subsequent steps.
2. Carry out end repair and addition of A at 3' as described below.
   1. Repair the damaged ends of FFPE DNA with enzymes to form flat ends, ensuring 3'-OH and 5'-P. Add A to the flattened double-stranded 3' ends.
   2. Prepare ERA mix by adding 7 µL of end repair buffer and 3 µL of end repair enzyme, gently manually flick 3x-5x, and centrifuge for 1-3 s.
   3. Add ERA mix to 50 µL of broken DNA sample, gently mix, and centrifuge for 1-3 s.
   4. Place it in the PCR instrument and select the ERA program (20 °C for 30 min; 65 °C for 30 min; 4 °C Hold; Lid 85 °C). Perform the next operation within 2 h.
3. Perform adapter ligation as described below.
   1. Prepare the reaction system LIG mix by adding 30 µL of ligation buffer, 10 µL of DNA ligase, and 10 µL of DNA adapter. Then, lightly manually flick 3x-5x and centrifuge for 1-3 s.
   2. Add 50 µL of LIG mix to the 60 µL product from the previous step, gently mix, then centrifuge for 1-3 s.
   3. Place the mixture in a PCR instrument, then select the LIG program (20 °C for 15 min without heating the lid; 70 °C for 10 min (heating cover); 4 °C Hold; Lid 85 °C).
4. Perform magnetic bead purification.
   1. Take 1.5 mL of a low adsorption centrifuge tube and add 88 µL of homogenized magnetic beads and 110 µL of adapter-ligated product. Vortex and mix well, and incubate at room temperature for 5 min.
   2. Immediately centrifuge the incubated sample for 1-3 s and place the centrifuge tube on a magnetic rack. Wait for the solution to clear (about 3-5 min).
   3. With the tube secured on the magnet, open the cap and carefully aspirate the cleared supernatant using a pipette without disturbing the bead pellet.
   4. Keep the tube on the magnet and add 300 µL of freshly prepared 75% ethanol to each tube.
   5. Wait for 1 min to allow the magnetic beads to fully settle. Slowly rotate the centrifuge tube horizontally once to absorb ethanol.
   6. Repeat steps 2.4.4 and 2.4.5 once, for a total of 2x.
   7. Perform instantaneous centrifugation for 1-3 s, return the tube to the magnetic rack and wait for 30 s. Use a pipette to remove any residual ethanol and keep the tube cover open.
   8. Dry the magnetic beads at room temperature for 2 minutes until the bead surface appears matte and shows no cracks.
   9. Elute the ligation product with 28 µL of elution buffer (EB).
      NOTE: Magnetic beads need to be equilibrated at room temperature for at least 30 min before use.
5. Amplify and purify the pre-library as described below.
   1. Prepare reaction system pre-mix by adding 10 µL of PCR buffer, 10 µL of PCR primers, 1.5 µL of dNTP mix, 1 µL of PCR enzyme. Gently manually tap 3x-5x and centrifuge for 1-3 s.
   2. Add 22.5 µL of pre-mix to 27.5 µL of the purified product from the previous step, gently mix, then centrifuge for 1-3 s.
   3. Select the program PRE: 98 °C 45 s; (98 °C 15 s, 60 °C 30 s, 72 °C 30 s) 12 cycles; 72 °C 2 min; 4 °C Hold; Lid 105 °C.
   4. After the PCR program is completed, perform magnetic bead purification using freshly prepared 75% ethanol. Elute the product with 18 µL of EB.
6. Capture hybrid as described below.
   1. Prepare reaction system A by adding 15 µL of pre-library and 4 µL of blocker mix.
   2. Place on PCR and run PCR program HYB (95 °C for 5 min; 65 °C Hold; Lid 105 °C).
   3. Prepare reaction system B HYB mix by adding 10 µL of hybridization buffer, 0.5 µL of RNase inhibitor blocker, and 1 µL of probe. Gently flick 3x-5x and centrifuge for 1-3 s.
      NOTE: The probe name is LCOO1, and the probe genes include EGFR, KRAS, BRAF, ALK, etc.
   4. When the temperature of the PCR instrument drops to 65 °C, add 11.5 µL of HYB mix to reaction system A, gently mix, then cover the PCR instrument lid and continue incubation for 16-24 h.
   5. Incubate wash buffer 2 in a 15 mL conical tube with a metal heater at 65 °C at a dosage of 600 µL per sample.
   6. Retrieve SCB magnetic beads, invert and mix 5x, vortex for 10 s, and equilibrate at room temperature for 30 min. Vortex for 10 s, transfer 25 µL to a 1.5 mL low adsorption centrifuge tube, place on a magnetic rack for 3 min, and discard the supernatant.
   7. Add 200 µL of Beads Wash Buffer to the 25 µL SCB magnetic beads, vortex and mix for 3 s, centrifuge momentarily for 1-3 s, let stand on the magnetic rack for 3 min, and discard the supernatant.
   8. Repeat step 2.6.7 2x, for a total of three times.
   9. Add 200 µL of beads wash buffer to the magnetic beads, vortex and mix for 3 s, resuspend for later use.
   10. Transfer approximately 30 µL of the hybrid sample to resuspended SCB magnetic beads, vortex and mix, place on a rotating mixer, incubate at room temperature for 30 min, and centrifuge for 1-3 s.
   11. Place the mixture tube on a magnetic rack for 3 min, then aspirate approximately 230 µL of supernatant. Add 500 µL of Wash buffer 1, vortex for 5 s, place on a rotary mixer, then incubate at room temperature for 15 min. Instantaneous centrifuge for 1-3 s.
   12. Place the magnetic bead sample mixture tube on a magnetic rack and let it stand for 3 min. Then, aspirate approximately 500 µL of supernatant and residual liquid.
   13. Preheat 150 µL of wash buffer 2 to 65 °C, vortex for 10 s, place on a constant temperature mixer, shake, and incubate at 65 °C for 10 min at a speed of 600 rpm. Perform instantaneous centrifugation for 1-3 s, place on a magnetic rack for 3 min, and aspirate approximately 150 µL of supernatant and residual liquid.
   14. Repeat step 2.6.13 3x, for a total of four times.
   15. Instantaneously centrifuge for 1-3 s to remove any residual liquid from the sample tube. Add 20 µL EB, mix up and down 8x-10x, mix well, and place on ice for later use.
7. Preparation and purification of the final library.
   1. Prepare the reaction system POST mix by adding 25 µL of amplification mix, 5 µL of index, and 20 µL of magnetic bead library. Gently tap 3x-5x and centrifuge for 1-3 s. Gently mix, centrifuge for 1-3 s.
   2. Choose program POST: 98 °C 45 s; (98 °C 15 s, 60 °C 30 s, 72 °C 30 s) 12 cycles; 72 °C 10 min; 4 °C Hold; Lid 105 °C.
   3. After the PCR program is completed, perform magnetic bead purification using freshly prepared 75% ethanol. Elute the final library with 20 µL of EB.

3. Fully automated library preparation (Table 5)

1. Press the Power button to activate the system and wait for initialization. Navigate to Program Setting and select Run Protocol, then select Protocol: Burning Rock HS.
2. Set the aliquot QC parameters as Sample Type: High Quality DNA; Input Amount (ng): 50; Pre PCR Cycles: 12; Post PCR Cycles: 12. Click RUN.
3. Insert prepackaged reagent cartridge into the designated slot (automatically validates reagent placement and status).
4. After loading samples, press Start to make the system run autonomously. Observe LED indicators to monitor progress.
5. After the program ends, click OK and the program will automatically transfer the final library into the library tube, finalizing the library preparation.

4. Sequencing and QC

1. Perform library quantification and QC as described below.
   1. Measure the pre-library and total library concentration using fluorometry.
   2. Detect the fragment size of the library. Use a kit to assess the fragment size of the library and the presence of adapters (Table of Materials).
      NOTE: If the final library contains primer dimers, an additional purification step must be added before mixing the samples on the machine. The qualified standard for inserting fragments is ≥ 150 bp, and the expected capture efficiency is ≥ 40 %.
   3. Dilute the sequencing library to 1.6 pM and 1300 µL for sequencing operations.
2. Carry out sequencing environment monitoring as described below.
   1. Control the sequencing environment, including indoor temperature (19-25 °C), indoor humidity (20%-80%).
   2. Complete instrument output data QC, including base above Q30, and cluster density passing filter (PF).

5. Sequencing data analysis and QC

1. Perform raw data processing and molecular profiling as described below.
   1. Use Burrows-Wheeler Aligner (version 0.7.10) to align sequencing data to hg19.
   2. Perform local alignment, optimization, duplication marking, and variant calling using Genome Analysis Tool Kit (version 3.2) and Vardict (version 1.5.1).
   3. Filter variants using the VarScan (version 2.4.3) fpfilter pipeline and remove loci with depths less than 50. Classify and exclude variants with population frequency over 0.1% in the ExAC, 1000 Genomes, dbSNP, or ESP6500SI-V2 databases as single nucleotide polymorphisms (SNPs).
   4. Annotate the remaining variants using SnpEff (version 3.6).
   5. Analyze Structural variants (SVs), including large genomic rearrangements (LGRs), using MarkSV (Burning Rock algorithm tool). Analyze copy number variants (CNVs) based on the depth of coverage data of capture intervals.
   6. Correct coverage data for sequencing biases resulting from guanine-cytosine content and probe design.
2. Assess sample quality, including mean depth, median depth, unique depth, insert size, lib complexity, coverage, Q30 ratio, and paired sample correlation ratio as described in²⁵.
3. For report generation, verify pipeline version and parameters to complete bioinformatics review. Use relevant guidelines to evaluate the pathogenicity of mutations^26,27. Issue final report.

Results

Utilizing an automated NGS analysis workflow with stringent quality controls, the laboratory performed comprehensive genomic profiling of NSCLC specimens. The optimized detection workflow (Figure 2) reliably identified several clinically actionable genomic alterations in representative tumor samples (Figure 3).

The sequencing results demonstrated a high-frequency EGFR p.L858R missense mutation (AF=89.25%) accompanied by signi...

Discussion

Every qualified NGS report lies behind a rigorous process, which remains complex and time-consuming²⁸. To address these issues, the laboratory developed a fully automated NGS system. This operation significantly reduces manual intervention, minimizes experimental errors, and substantially shortens turnaround time. Therefore, this system has provided a more reliable and efficient strategy for NGS, establishing a new thought for data quality in genomic research.

The inte...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agilent  High Sensitivity DNA Kit	Agilent	5067-4626	For use with the  Agilent 2100 Bioanalyzer System
Agilent DNA 1000 Reagents Kit	Agilent	5067-1504	For use with the  Agilent 2100 Bioanalyzer System
Agilent 2100 Bioanalyzer	Agilent	G2939A	Assess the fragment size of the library and the presence of adapters.
Concert Fully automatic nucleic acid purification instrument	CONCERT	MC1002	Used for DNA extraction
Equalbit 1×dsDNA HS Assay Kit	Vazyme	EQ121-02-AA	For use with the  Qubit 3.0 Fluorometer
Human EGRR/KRAS/BRAF/ALK gene mutation combined detection kit	Burning Rock	BR-LC00102	library construction
Magnis BR	Agilent Technologies	G7595AA	Automated NGS library preparation
ME220 Focused-ultrasonicator	Covaris	500396	ultrasonication
NanoDrop One/OneC	Thermo Fisher	ND-ONE-W	Full wavelength UV visible spectrophotometric detection
NextSeq 550Dx	Illumina	20005715	sequence
NSQ 500/550 Hi Output KT v2.5(300CYS)	Illumina	20024908	For use with the NextSeq 550Dx
Nucleic acid extraction reagent	CONCERT	RC1102	Used for DNA extraction
Qubit 3.0 Fluorometer	Life	Q33216	Used for nucleic acid or library concentration detection
Qubit dsDNA HS Assay Kit	Life	Q32851	For use with the  Qubit 3.0 Fluorometer