Name: Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing (Video) | JoVE Journal
Uploaded: 2025-10-10T14:25:00.000+00:00
Duration: 5 min 17 s
Description: Discover an automated next-generation sequencing workflow for detecting targetable genomic alterations in non-small cell lung cancer using formalin-fixed paraffin-embedded tissues. This ISO15189-accredited method improves diagnostic capabilities.

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5 minutes

This study was supported by grants from the Beijing Natural Science Foundation of the People&#39;s Republic of China (7252122), Beijing Xisike Clinical Oncology Research Foundation of the People&#39;s Republic of China (Y-HS202402-0021), the Residency Training Teaching Research Fund of Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (E2025004), and the National High Level Hospital Clinical Research Funding (2025-LYZX-R-B03).

The protocol received an IRB waiver for anonymized archival samples.
1. Pre-testing steps and QC
<ol>
	<li>Register sample information in the laboratory information management system (Table 1). Fill out the informed consent form for gene mutation testing.</li>
	<li>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.</li>
	<li>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 (&#8805; 0.5 cm x 0.5 cm) 6-10 &#181;L, five sheets; small sample size (&#60; 0.5 cm x 0.5 cm) and puncture samples, 6-10 &#181;L, 10 sheets.</li>
	<li>Perform routine hematoxylin-eosin (HE) staining to evaluate tumor cell content.</li>
	<li>Select samples containing &#8805;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.</li>
	<li>Perform nucleic acid extraction using an automated purification system as described below.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Refer to the instructions of the fully automatic nucleic acid purifier and nucleic acid extraction reagent to extract nucleic acid.</li>
		<li>Then, select the C1102 Program, choose sample dewaxing incubation time as 16 h, and elution volume as 100 &#181;L. 
		NOTE: If the extracted DNA solution is not immediately tested, it must be stored at -20 &#176;C for no more than 6 months.</li>
	</ol>
	</li>
	<li>Assess nucleic acid quality (Table 2) as described below.
	<ol>
		<li>Use agarose gel electrophoresis to determine DNA fragment size. Use Agilent2100 and other microfluidic capillary electrophoresis analysis systems for low-input samples.</li>
		<li>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 &#176;C.</li>
		<li>Connect to the positive electrode (red) on the electrophoresis instrument. Perform negative pole (black) to voltage 180 V, run for 15 min21,22.</li>
		<li>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.</li>
		<li>Use spectrophotometry to verify purity. Ensure a OD260/OD280 &#62; 1.8; OD260/OD230 &#62; 2.0 to indicate acceptable contaminant levels23,24.</li>
		<li>Use detection kit (Table of Materials) based on sequence-specific fluorescent dyes for fluorometric assay to precisely measure DNA concentration.</li>
		<li>Add 1 &#181;L of DNA to be tested to 199 &#181;L of working solution, then mix, briefly centrifuge, and measure its concentration using a fluorometer (Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Library preparation and QC (Figure 1)
<ol>
	<li>Prepare fragment genomic DNA using ultrasonication (Table of Materials) as described below.
	<ol>
		<li>Mix 200 ng genomic DNA in 50 &#181;L of low Tris-EDTA (TE) buffer. Then, keep the sample tube in a pre-cooled (8 &#176;C) sample rack.</li>
		<li>Set instrument parameter according to Table 4. Then initiate fragmentation.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Carry out end repair and addition of A at 3&#39; as described below.
	<ol>
		<li>Repair the damaged ends of FFPE DNA with enzymes to form flat ends, ensuring 3&#39;-OH and 5&#39;-P. Add A to the flattened double-stranded 3&#39; ends.</li>
		<li>Prepare ERA mix by adding 7 &#181;L of end repair buffer and 3 &#181;L of end repair enzyme, gently manually flick 3x-5x, and centrifuge for 1-3 s.</li>
		<li>Add ERA mix to 50 &#181;L of broken DNA sample, gently mix, and centrifuge for 1-3 s.</li>
		<li>Place it in the PCR instrument and select the ERA program (20 &#176;C for 30 min; 65 &#176;C for 30 min; 4 &#176;C Hold; Lid 85 &#176;C). Perform the next operation within 2 h.</li>
	</ol>
	</li>
	<li>Perform adapter ligation as described below.
	<ol>
		<li>Prepare the reaction system LIG mix by adding 30 &#181;L of ligation buffer, 10 &#181;L of DNA ligase, and 10 &#181;L of DNA adapter. Then, lightly manually flick 3x-5x and centrifuge for 1-3 s.</li>
		<li>Add 50 &#181;L of LIG mix to the 60 &#181;L product from the previous step, gently mix, then centrifuge for 1-3 s.</li>
		<li>Place the mixture in a PCR instrument, then select the LIG program (20 &#176;C for 15 min without heating the lid; 70 &#176;C for 10 min (heating cover); 4 &#176;C Hold; Lid 85 &#176;C).</li>
	</ol>
	</li>
	<li>Perform magnetic bead purification.
	<ol>
		<li>Take 1.5 mL of a low adsorption centrifuge tube and add 88 &#181;L of homogenized magnetic beads and 110 &#181;L of adapter-ligated product. Vortex and mix well, and incubate at room temperature for 5 min.</li>
		<li>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).</li>
		<li>With the tube secured on the magnet, open the cap and carefully aspirate the cleared supernatant using a pipette without disturbing the bead pellet.</li>
		<li>Keep the tube on the magnet and add 300 &#181;L of freshly prepared 75% ethanol to each tube.</li>
		<li>Wait for 1 min to allow the magnetic beads to fully settle. Slowly rotate the centrifuge tube horizontally once to absorb ethanol.</li>
		<li>Repeat steps 2.4.4 and 2.4.5 once, for a total of 2x.</li>
		<li>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.</li>
		<li>Dry the magnetic beads at room temperature for 2 minutes until the bead surface appears matte and shows no cracks.</li>
		<li>Elute the ligation product with 28 &#181;L of elution buffer (EB). 
		NOTE: Magnetic beads need to be equilibrated at room temperature for at least 30 min before use.</li>
	</ol>
	</li>
	<li>Amplify and purify the pre-library as described below.
	<ol>
		<li>Prepare reaction system pre-mix by adding 10 &#181;L of PCR buffer, 10 &#181;L of PCR primers, 1.5 &#181;L of dNTP mix, 1 &#181;L of PCR enzyme. Gently manually tap 3x-5x and centrifuge for 1-3 s.</li>
		<li>Add 22.5 &#181;L of pre-mix to 27.5 &#181;L of the purified product from the previous step, gently mix, then centrifuge for 1-3 s.</li>
		<li>Select the program PRE: 98 &#176;C 45 s; (98 &#176;C 15 s, 60 &#176;C 30 s, 72 &#176;C 30 s) 12 cycles; 72 &#176;C 2 min; 4 &#176;C Hold; Lid 105 &#176;C.</li>
		<li>After the PCR program is completed, perform magnetic bead purification using freshly prepared 75% ethanol. Elute the product with 18 &#181;L of EB.</li>
	</ol>
	</li>
	<li>Capture hybrid as described below.
	<ol>
		<li>Prepare reaction system A by adding 15 &#181;L of pre-library and 4 &#181;L of blocker mix.</li>
		<li>Place on PCR and run PCR program HYB (95 &#176;C for 5 min; 65 &#176;C Hold; Lid 105 &#176;C).</li>
		<li>Prepare reaction system B HYB mix by adding 10 &#181;L of hybridization buffer, 0.5 &#181;L of RNase inhibitor blocker, and 1 &#181;L 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.</li>
		<li>When the temperature of the PCR instrument drops to 65 &#176;C, add 11.5 &#181;L of HYB mix to reaction system A, gently mix, then cover the PCR instrument lid and continue incubation for 16-24 h.</li>
		<li>Incubate wash buffer 2 in a 15 mL conical tube with a metal heater at 65 &#176;C at a dosage of 600 &#181;L per sample.</li>
		<li>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 &#181;L to a 1.5 mL low adsorption centrifuge tube, place on a magnetic rack for 3 min, and discard the supernatant.</li>
		<li>Add 200 &#181;L of Beads Wash Buffer to the 25 &#181;L 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.</li>
		<li>Repeat step 2.6.7 2x, for a total of three times.</li>
		<li>Add 200 &#181;L of beads wash buffer to the magnetic beads, vortex and mix for 3 s, resuspend for later use.</li>
		<li>Transfer approximately 30 &#181;L 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.</li>
		<li>Place the mixture tube on a magnetic rack for 3 min, then aspirate approximately 230 &#181;L of supernatant. Add 500 &#181;L 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.</li>
		<li>Place the magnetic bead sample mixture tube on a magnetic rack and let it stand for 3 min. Then, aspirate approximately 500 &#181;L of supernatant and residual liquid.</li>
		<li>Preheat 150 &#181;L of wash buffer 2 to 65 &#176;C, vortex for 10 s, place on a constant temperature mixer, shake, and incubate at 65 &#176;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 &#181;L of supernatant and residual liquid.</li>
		<li>Repeat step 2.6.13 3x, for a total of four times.</li>
		<li>Instantaneously centrifuge for 1-3 s to remove any residual liquid from the sample tube. Add 20 &#181;L EB, mix up and down 8x-10x, mix well, and place on ice for later use.</li>
	</ol>
	</li>
	<li>Preparation and purification of the final library.
	<ol>
		<li>Prepare the reaction system POST mix by adding 25 &#181;L of amplification mix, 5 &#181;L of index, and 20 &#181;L of magnetic bead library. Gently tap 3x-5x and centrifuge for 1-3 s. Gently mix, centrifuge for 1-3 s.</li>
		<li>Choose program POST: 98 &#176;C 45 s; (98 &#176;C 15 s, 60 &#176;C 30 s, 72 &#176;C 30 s) 12 cycles; 72 &#176;C 10 min; 4 &#176;C Hold; Lid 105 &#176;C.</li>
		<li>After the PCR program is completed, perform magnetic bead purification using freshly prepared 75% ethanol. Elute the final library with 20 &#181;L of EB.</li>
	</ol>
	</li>
</ol>
3. Fully automated library preparation (Table 5)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Insert prepackaged reagent cartridge into the designated slot (automatically validates reagent placement and status).</li>
	<li>After loading samples, press Start to make the system run autonomously. Observe LED indicators to monitor progress.</li>
	<li>After the program ends, click OK and the program will automatically transfer the final library into the library tube, finalizing the library preparation.</li>
</ol>
4. Sequencing and QC
<ol>
	<li>Perform library quantification and QC as described below.
	<ol>
		<li>Measure the pre-library and total library concentration using fluorometry.</li>
		<li>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 &#8805; 150 bp, and the expected capture efficiency is &#8805; 40 %.</li>
		<li>Dilute the sequencing library to 1.6 pM and 1300 &#181;L for sequencing operations.</li>
	</ol>
	</li>
	<li>Carry out sequencing environment monitoring as described below.
	<ol>
		<li>Control the sequencing environment, including indoor temperature (19-25 &#176;C), indoor humidity (20%-80%).</li>
		<li>Complete instrument output data QC, including base above Q30, and cluster density passing filter (PF).</li>
	</ol>
	</li>
</ol>
5. Sequencing data analysis and QC
<ol>
	<li>Perform raw data processing and molecular profiling as described below.
	<ol>
		<li>Use Burrows-Wheeler Aligner (version 0.7.10) to align sequencing data to hg19.</li>
		<li>Perform local alignment, optimization, duplication marking, and variant calling using Genome Analysis Tool Kit (version 3.2) and Vardict (version 1.5.1).</li>
		<li>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).</li>
		<li>Annotate the remaining variants using SnpEff (version 3.6).</li>
		<li>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.</li>
		<li>Correct coverage data for sequencing biases resulting from guanine-cytosine content and probe design.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>For report generation, verify pipeline version and parameters to complete bioinformatics review. Use relevant guidelines to evaluate the pathogenicity of mutations26,27. Issue final report.</li>
</ol>

<ol><li>Feng, R. M., Zong, Y. N., Cao, S. M., Xu, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+cancer+situation+in+China%3A+Good+or+bad+news+from+the+2018+global+cancer+statistics%5BTitle%5D)+AND+%22Cancer+Commun+%28Lond%29%22%5BJournal%5D)">Current cancer situation in China: Good or bad news from the 2018 global cancer statistics.</a> Cancer Commun (Lond). 39 (1), 22(2019).</li>
<li>Siegel, R. L., Kratzer, T. B., Giaquinto, A. N., Sung, H., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+statistics%2C+2025%5BTitle%5D)+AND+%22CA+Cancer+J+Clin%22%5BJournal%5D)">Cancer statistics, 2025.</a> CA Cancer J Clin. 75 (1), 10-45 (2025).</li>
<li>Sung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+cancer+statistics+2020%3A+Globocan+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries%5BTitle%5D)+AND+%22CA+Cancer+J+Clin%22%5BJournal%5D)">Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA Cancer J Clin. 71 (3), 209-249 (2021).</li>
<li>Fukuoka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-institutional+randomized+phase+II+trial+of+gefitinib+for+previously+treated+patients+with+advanced+non-small-cell+lung+cancer%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer.</a> J Clin Oncol. 41 (6), 1162-1171 (2023).</li>
<li>Kris, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+gefitinib%2C+an+inhibitor+of+the+epidermal+growth+factor+receptor+tyrosine+kinase%2C+in+symptomatic+patients+with+non-small+cell+lung+cancer%3A+A+randomized+trial%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: A randomized trial.</a> JAMA. 290 (16), 2149-2158 (2003).</li>
<li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erlotinib+versus+chemotherapy+as+first-line+treatment+for+patients+with+advanced+EGFR+mutation-positive+non-small-cell+lung+cancer+%28OPTIMAL%2C+CTONG-0802%29%3A+A+multicentre%2C+open-label%2C+randomised%2C+phase+3+study%5BTitle%5D)+AND+%22Lancet+Oncol%22%5BJournal%5D)">Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): A multicentre, open-label, randomised, phase 3 study.</a> Lancet Oncol. 12 (8), 735-742 (2011).</li>
<li>Wu, K., House, L., Liu, W., Cho, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Personalized+targeted+therapy+for+lung+cancer%5BTitle%5D)+AND+%22Zhongguo+Fei+Ai+Za+Zhi%22%5BJournal%5D)">Personalized targeted therapy for lung cancer.</a> Zhongguo Fei Ai Za Zhi. 16 (8), C21-C34 (2013).</li>
<li>Sharma, S. V., Settleman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oncogenic+shock%3A+Turning+an+activated+kinase+against+the+tumor+cell%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Oncogenic shock: Turning an activated kinase against the tumor cell.</a> Cell Cycle. 5 (24), 2878-2880 (2006).</li>
<li>Kumar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+early+growth+response+1+%28EGR1%29+expression+correlates+with+resistance+to+anti-EGFR+treatment+in+vitro+and+with+poorer+outcome+in+metastatic+colorectal+cancer+patients+treated+with+cetuximab%5BTitle%5D)+AND+%22Clin+Transl+Oncol%22%5BJournal%5D)">High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.</a> Clin Transl Oncol. 19 (6), 718-726 (2017).</li>
<li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Status+of+10+targeted+genes+of+non-small+cell+lung+cancer+in+Eastern+China%3A+A+study+of+884+patients+based+on+NGS+in+a+single+institution%5BTitle%5D)+AND+%22Thorac+Cancer%22%5BJournal%5D)">Status of 10 targeted genes of non-small cell lung cancer in Eastern China: A study of 884 patients based on NGS in a single institution.</a> Thorac Cancer. 11 (9), 2580-2589 (2020).</li>
<li>Wen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+signature+of+driver+genes+identified+by+target+next-generation+sequencing+in+Chinese+non-small+cell+lung+cancer%5BTitle%5D)+AND+%22Oncologist%22%5BJournal%5D)">Genomic signature of driver genes identified by target next-generation sequencing in Chinese non-small cell lung cancer.</a> Oncologist. 24 (11), e1070-e1081 (2019).</li>
<li>Tan, N., Li, Y., Ying, J., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histological+transformation+in+lung+adenocarcinoma%3A+Insights+of+mechanisms+and+therapeutic+windows%5BTitle%5D)+AND+%22J+Transl+Int+Med%22%5BJournal%5D)">Histological transformation in lung adenocarcinoma: Insights of mechanisms and therapeutic windows.</a> J Transl Int Med. 12 (5), 452-465 (2024).</li>
<li>Coco, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next+generation+sequencing+in+non-small+cell+lung+cancer%3A+New+avenues+toward+personalized+medicine%5BTitle%5D)+AND+%22Curr+Drug+Targets%22%5BJournal%5D)">Next generation sequencing in non-small cell lung cancer: New avenues toward personalized medicine.</a> Curr Drug Targets. 16 (1), 47-59 (2015).</li>
<li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+and+cellular+choreography+of+early+mammalian+lung+development%5BTitle%5D)+AND+%22Med+Rev%22%5BJournal%5D)">The molecular and cellular choreography of early mammalian lung development.</a> Med Rev. 4 (3), 192-206 (2024).</li>
<li>Wang, R., Wang, G., Zhang, N., Li, X., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+evaluation+and+cost-effectiveness+analysis+of+serum+tumor+markers+in+lung+cancer%5BTitle%5D)+AND+%22Biomed+Res+Int%22%5BJournal%5D)">Clinical evaluation and cost-effectiveness analysis of serum tumor markers in lung cancer.</a> Biomed Res Int. 2013, 195692(2013).</li>
<li>McGinn, S., Gut, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+sequencing-spanning+the+generations%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">DNA sequencing-spanning the generations.</a> Nat Biotechnol. 30 (4), 366-372 (2013).</li>
<li>Larson, N. B., Oberg, A. L., Adjei, A. A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+clinician%27s+guide+to+bioinformatics+for+next-generation+sequencing%5BTitle%5D)+AND+%22J+Thorac+Oncol%22%5BJournal%5D)">A clinician's guide to bioinformatics for next-generation sequencing.</a> J Thorac Oncol. 18 (2), 143-157 (2023).</li>
<li>McCombie, W. R., McPherson, J. D., Mardis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+sequencing+technologies%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Med%22%5BJournal%5D)">Next-generation sequencing technologies.</a> Cold Spring Harb Perspect Med. 9 (11), a036798(2019).</li>
<li>Van Dijk, E. L., Jaszczyszyn, Y., Thermes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Library+preparation+methods+for+next-generation+sequencing%3A+Tone+down+the+bias%5BTitle%5D)+AND+%22Exp+Cell+Res%22%5BJournal%5D)">Library preparation methods for next-generation sequencing: Tone down the bias.</a> Exp Cell Res. 322 (1), 12-20 (2014).</li>
<li>Rao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+unreliability+of+ALK+variant+allele+frequency+in+the+efficacy+prediction+of+targeted+therapy+in+NSCLC%5BTitle%5D)+AND+%22Front+Med%22%5BJournal%5D)">Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC.</a> Front Med. 17 (3), 493-502 (2023).</li>
<li>Koontz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarose+gel+electrophoresis%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Agarose gel electrophoresis.</a> Methods Enzymol. 529, 35-45 (2013).</li>
<li>Holmes, D. L., Stellwagen, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoresis+of+DNA+in+oriented+agarose+gels%5BTitle%5D)+AND+%22J+Biomol+Struct+Dyn%22%5BJournal%5D)">Electrophoresis of DNA in oriented agarose gels.</a> J Biomol Struct Dyn. 7 (2), 311-327 (1989).</li>
<li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+control+of+next-generation+sequencing-based+in+vitro+diagnostic+test+for+onco-relevant+mutations+using+multiplex+reference+materials+in+plasma%5BTitle%5D)+AND+%22J+Cancer%22%5BJournal%5D)">Quality control of next-generation sequencing-based in vitro diagnostic test for onco-relevant mutations using multiplex reference materials in plasma.</a> J Cancer. 9 (9), 1680-1688 (2018).</li>
<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+NGS+library+construction+approach+using+DNA+isolated+from+human+cancer+formalin-fixed+paraffin-embedded+samples%5BTitle%5D)+AND+%22Anat+Rec+%28Hoboken%29%22%5BJournal%5D)">An improved NGS library construction approach using DNA isolated from human cancer formalin-fixed paraffin-embedded samples.</a> Anat Rec (Hoboken). 302 (6), 941-946 (2019).</li>
<li>Wood, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National+comprehensive+cancer+network+%28NCCN%29+clinical+practice+guidelines+for+lung+cancer+screening%5BTitle%5D)+AND+%22Thorac+Surg+Clin%22%5BJournal%5D)">National comprehensive cancer network (NCCN) clinical practice guidelines for lung cancer screening.</a> Thorac Surg Clin. 25 (2), 185-197 (2015).</li>
<li>Chinese Society of Pathology Quality Control, Chinese Medical Association Chinese Society of Oncology, China Anti-Cancer Association Chinese Society of Lung Cancer, Chinese Thoracic Oncology Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+on+clinical+practice+of+molecular+tests+in+non-small+cell+lung+cancer+in+China%5BTitle%5D)+AND+%22Zhonghua+Bing+Li+Xue+Za+Zhi%22%5BJournal%5D)">Guidelines on clinical practice of molecular tests in non-small cell lung cancer in China.</a> Zhonghua Bing Li Xue Za Zhi. 50 (4), 323-332 (2021).</li>
<li>Rao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+an+effective+quality+evaluation+strategy+of+next-generation+sequencing+for+accurate+detecting+non-small+cell+lung+cancer+samples+with+variable+characteristics%3A+A+real-world+clinical+practice%5BTitle%5D)+AND+%22J+Cancer+Res+Clin+Oncol%22%5BJournal%5D)">Developing an effective quality evaluation strategy of next-generation sequencing for accurate detecting non-small cell lung cancer samples with variable characteristics: A real-world clinical practice.</a> J Cancer Res Clin Oncol. 149 (8), 4889-4897 (2023).</li>
<li>Hutchins, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Practical+guidance+to+implementing+quality+management+systems+in+public+health+laboratories+performing+next-generation+sequencing%3A+Personnel%2C+equipment%2C+and+process+management+%28Phase+1%29%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Practical guidance to implementing quality management systems in public health laboratories performing next-generation sequencing: Personnel, equipment, and process management (Phase 1).</a> J Clin Microbiol. 57 (8), e00261-e00319 (2019).</li>
<li>Haile, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+high+throughput+nucleic+acid+purification+from+formalin-fixed+paraffin-embedded+tissue+samples+for+next+generation+sequence+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis.</a> PLoS One. 12 (6), e0178706(2017).</li>
<li>Larguinho, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+fast+and+efficient+ultrasonic-based+strategy+for+DNA+fragmentation%5BTitle%5D)+AND+%22Talanta%22%5BJournal%5D)">Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation.</a> Talanta. 81 (3), 881-886 (2010).</li>
<li>Xuan, J., Yu, Y., Qing, T., Guo, L., Shi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+sequencing+in+the+clinic%3A+Promises+and+challenges%5BTitle%5D)+AND+%22Cancer+Lett%22%5BJournal%5D)">Next-generation sequencing in the clinic: Promises and challenges.</a> Cancer Lett. 340 (2), 284-295 (2013).</li>
<li>Senst, A., Bonsiepe, H., Kron, S., Schulz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+the+Agilent+2100+bioanalyzer+instrument+as+quality+control+for+next-generation+sequencing%5BTitle%5D)+AND+%22J+Forensic+Sci%22%5BJournal%5D)">Application of the Agilent 2100 bioanalyzer instrument as quality control for next-generation sequencing.</a> J Forensic Sci. 69 (6), 2192-2196 (2024).</li>
<li>Li, W., Qiu, T., Ling, Y., Gao, S., Ying, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subjecting+appropriate+lung+adenocarcinoma+samples+to+next-generation+sequencing-based+molecular+testing%3A+Challenges+and+possible+solutions%5BTitle%5D)+AND+%22Mol+Oncol%22%5BJournal%5D)">Subjecting appropriate lung adenocarcinoma samples to next-generation sequencing-based molecular testing: Challenges and possible solutions.</a> Mol Oncol. 12 (5), 677-689 (2018).</li>
<li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+unreliability+of+uncommon+ALK%2C+ROS1%2C+and+RET+genomic+breakpoints+in+predicting+the+efficacy+of+targeted+therapy+in+NSCLC%5BTitle%5D)+AND+%22J+Thorac+Oncol%22%5BJournal%5D)">Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.</a> J Thorac Oncol. 16 (3), 404-418 (2021).</li>
</ol>

The authors have no conflicts of interest to disclose.

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...

Non-small cell lung cancer (NSCLC) accounts for 75%-85% of all lung cancer cases, representing both a clinical priority and a therapeutic challenge1,2,3. 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 therapies4,5,6. Targeted therapy offers precise efficacy, high specificity, and minimal side effects7,8. 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 application9,10,11,12.
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 alterations13,14. Meanwhile, NGS achieves comprehensive testing in a single run, relatively reducing cost, sample consumption, and testing turnaround time15,16. 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.

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

detection of targetable alterations in non-small cell lung cancer using next-generation sequencing

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

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 (&#8805;20 %), DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues (DNA input &#8805; 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.

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...

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.

The success of targeted therapy in non-small cell lung cancer (NSCLC) hinges on the precise identification of driver alterations, including mutations, ...

My research discovers genetic mutations in cancer to improve diagnosis and treatment. To begin, register the sample information in the Laboratory Information Management System and fill out the informed consent form for gene mutation testing. Using disposable microtome blades, cut sections from the non-small cell lung cancer, formal and fixed paraffin embedded sample.Then perform routine hematoxylin and eosin staining to evaluate the tumor cell content. For nucleic acid extraction, using a microtome, section the formal and fixed paraffin embedded block to obtain a ribbon, and place the ribbon into a 1.5-milliliter microcentrifuge tube. Transfer the tube to the designated sample input location on the automated extraction instrument.Next, use an automatic extraction instrument with an automatic extraction kit. Replace manual pipetting steps with an automatic magnetic bead nucleic acid extraction system. Then select the C1102 program on the instrument.Choose the sample de-waxing incubation time as 16 hours, and set the elucian volume to 100 microliters. Use spectrophotometry to verify DNA purity. Then use a detection kit with sequence-specific fluorescent dyes to perform a fluorometric assay for precise DNA concentration measurement.To prepare fragmented genomic DNA using ultrasonication, mix 200 nanograms of genomic DNA in 50 microliters of low tris-EDTA buffer. Place the sample tube in a pre-cooled sample rack, maintained at eight degrees Celsius. Set the instrument parameters according to the protocol and initiate DNA fragmentation.To start automated library preparation, press the power button to activate the system and wait for the initialization to complete. Navigate to program setting, select run protocol, and choose protocol BurningRock HS.Set the sample type to high quality DNA, input amount nanograms to 50, pre-PCR cycles to 12, and post-PCR cycles to 12. Then click Run to proceed.Then insert the prepackaged reagent cartridge into the designated slot of the instrument, which will automatically validate reagent placement and status. After loading the samples, press start to initiate the autonomous run. Observe the LED indicators on the system to monitor the progress.After the program ends, click OK to confirm and the system will automatically transfer the final library into the library tube, completing library preparation. Measure the concentration of both pre-library and total library using fluorometry. Dilute the sequencing library to 1.6 picomolar and 1300 microliters for sequencing operations.Control the sequencing environment by maintaining an indoor temperature and indoor humidity. Complete quality control of the instrument output data by verifying base quality above Q 30 and cluster density passing filter. The optimized detection workflow reliably identified several clinically actionable genomic alterations in representative tumor samples.The sequencing results demonstrated a high frequency EGFR p. L858R missense mutation, accompanied by a significant EGFR gene copy number amplification. Moderate amplification of the MET gene was observed and low level amplification of the BRAF gene was observed.The sample exhibited an intermediate tumor mutation burden and microsatellite stable status. All targeted gene loci, including ALK, BRAF, KRAS, and ROS1 achieved a 100%detection rate across four replicate experiments. Each mutation was consistently detected in all four repeated experiments, with expected abundance values remaining within the normal fluctuation range, NJS provides a compressive genetic profile for non-small cell lung cancer by detecting common, rare, and novel genes at the same time.Our future research will focus on combining molecular pathology with artificial intelligence to explore new biomarkers for lung cancer.

detection-targetable-alterations-non-small-cell-lung-cancer-using

Research

JoVE Journal

Cancer Research

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

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

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients’ tumors into mice and testing responses to corresponding drugs.

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

The increase of molecular biomarkers to be tested for non-squamous non-small cell lung cancer (NS-NSCLC) care management has prompted the development of fast and reliable molecular detection methods. We describe a workflow for genomic alteration assessment for NS-NSCLC patients using an ultra-fast-next generation sequencing (NGS) approach.

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Lung Tumor Cell Recruitment Assay

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

Discover an automated next-generation sequencing workflow for detecting targetable genomic alterations in non-small cell lung cancer using formalin-fixed paraffin-embedded tissues. This ISO15189-accredited method improves diagnostic capabilities.

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

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