What Is Next-Generation Sequencing (NGS)? Its Use in Oncology
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What is next-generation sequencing (NGS)? Its use in oncology
Next-generation sequencing (NGS) is a genetic analysis technology that divides DNA into millions of small fragments and reads all of them simultaneously . This allows hundreds of genes, or even an entire genome, to be sequenced in a few days using a single test. In oncology, NGS reveals a tumor's “genetic identity,” indicating which targeted drug or immunotherapy may work, detecting inherited cancer predisposition and allowing tumor DNA monitoring in blood samples through liquid biopsy. This article explains how NGS works, its types, cancer treatment applications, limitations and the situation in Türkiye.
- What does it do? It fragments DNA or RNA, reads millions of fragments in parallel and identifies changes by comparing them computationally with the reference human genome.
- Why is it important? Older methods examine genes individually, while NGS screens dozens or hundreds in a single test, efficiently using limited tissue.
- Main oncology applications: Targeted treatment selection, tumor-agnostic therapies such as NTRK, MSI-H and TMB-H, inherited cancer testing and liquid biopsy.
- Guidelines: The European Society for Medical Oncology (ESMO) recommends NGS in advanced non-small cell lung, prostate, ovarian and biliary tract cancers; the 2024 update added breast cancer and some rare tumors.
- Realistic expectations: An actionable alteration is found in approximately one-third of patients, but fewer obtain a matching treatment.
- Türkiye: Tissue- and blood-based NGS panels are included in the SGK reimbursement list (SUT), according to gene count.
Cancer develops through changes, or mutations, accumulating in cellular DNA over time. Two cancers originating in the same organ may differ greatly in these changes. For example, one lung cancer patient's tumor may contain an EGFR mutation, while another contains an ALK fusion, making their most effective treatments entirely different. We explained why the location of mutations within tumors and genes matters in cancer mutations in our related article.
NGS transformed oncology through its ability, rather than seeking these alterations individually, to examine them all together. Previously in lung cancer, first EGFR, then ALK, then ROS1 were tested, consuming separate tissue for each. Today, one NGS panel examines all these genes and even hundreds together.
Think of the human genome as a giant book containing approximately 3,1 billion letters . The older method, Sanger sequencing, resembles reading a specific page line by line: accurate but slow. NGS cuts many copies of the book into millions of tiny strips, reads them all simultaneously and finds typographical errors by comparing them computationally with a standard “reference book.” Because each line is read dozens or hundreds of times, a single reading error does not mislead, and even changes present in few cells can be detected.
Basic Concepts: Gene, Genome and Exome
- DNA and bases: DNA consists of a sequence of four building blocks, nucleotides, named by their bases' initials: A (adenine), T (thymine), G (guanine), C (cytosine). “Sequencing” means reading their order. We described the discovery of DNA's double helix in DNA double helix in our related article.
- Chromosome: Human cells contain 23 pairs, totaling 46 chromosomes (22 autosomal pairs and one pair of sex chromosomes).
- Genome: The complete DNA sequence in a cell. Approximately 3,1 billion base pairs in humans.
- Gene: A DNA region with a particular function. Current databases identify approximately 19.900 protein-coding genes in humans (the often-cited older figure of 25.000 is outdated), plus tens of thousands of RNA genes not translated into protein.
- Exome: The total protein-coding portions of genes, or exons. It represents approximately only %1,5 of the genome but contains a substantial proportion of known disease-causing changes.
- Somatic and germline (inherited) alterations: Somatic alterations occur only in tumor cells and are not inherited. Germline alterations are present from birth in all body cells and can run in families. NGS detects both; the sample examined, tumor or blood, determines the distinction.
Historical Context: Sequencing Firsts from 1977 to Today
We discussed the Human Genome Project's story and effects on cancer in detail in Human Genome Project our related article. The essence of this history: the first human genome required 13 years and billions of dollars, whereas hundreds of genes in a patient's tumor can now be examined in days at much lower cost.
How Is NGS Performed?
What distinguishes NGS is that sequencing reactions run in parallel : millions of DNA fragments are read at once. Short barcode sequences are added to each sample's fragments, allowing samples from multiple patients to be read together in one run, with computers assigning every read to the correct patient. This reduces cost per sample.
Figure 1 · NGS from Sample to Report
Laboratory and computational bioinformatics steps jointly determine the result.
The most widely used technology, Illumina, uses “sequencing by synthesis”: as a new strand forms opposite a single DNA strand, each added base carries a different-colored fluorescent label, recorded by a camera. Another technology, Ion Torrent, measures small pH changes accompanying each added base instead of light. More recently widespread long-read technologies, PacBio and Oxford Nanopore, read much longer fragments at once and resolve complex regions; they completed the first gapless human genome in 2022.
Depth: Why Read the Same Region Hundreds of Times?
In NGS, the number of times a region is read is called read depth or coverage. Depth determines reliability. For inherited variants, whole-genome sequencing recommends 30-50 reads per region and whole-exome sequencing approximately 100. Tumor testing requires much more because tumor samples mix normal cells and mutations may occur in only some tumor cells. FoundationOne CDx, for example, targets a median of more than 500 reads per region. Liquid biopsy depth is even higher because blood contains little tumor DNA; the FoundationOne Liquid CDx label reports a median detection limit of around %0,4 in its specifications.
Figure 2 · Read Depth and Variant Allele Frequency
The more often a region is read, the greater the chance of detecting a mutation present in few cells.
NGS Types: Panels, Exomes, Genomes, RNA and Liquid Biopsy
| Type | What does it read? | Typical depth | Oncology role |
|---|---|---|---|
| Targeted gene panel | Dozens to hundreds of selected genes | High (median 500+) | Standard in daily practice; for treatment selection |
| Whole-exome sequencing (WES) | Coding portions of all genes (%1,5 of the genome) | Approximately 100 | Inherited disease diagnosis and research |
| Whole-genome sequencing (WGS) | The entire genome | 30-50 (inherited) | Research; entering clinical use in some centers |
| RNA sequencing | RNA produced from genes | — | Fusion genes (ALK, ROS1, RET, NTRK, NRG1) and gene expression |
| Liquid biopsy (ctDNA) | Cell-free tumor DNA in blood | Very high | Insufficient tissue, resistance mutation monitoring (for example ESR1, EGFR T790M), residual disease research |
A common misconception is “NGS does not analyze the whole genome; examining the whole exome is the best way to screen mutations.” Whole-genome sequencing is also an NGS application. In oncology practice, the commonest method is neither exome nor genome sequencing, but reads genes known to matter in tumors at very high depth through targeted panels. Which method is “best” depends on the question.
What Can NGS Detect?
- Point mutations: A single-base change (for example EGFR L858R, KRAS G12C, BRAF V600E).
- Small insertions and deletions: Addition or loss of a few bases (for example EGFR exon 19 deletion, HER2 exon 20 insertions).
- Copy-number alterations: Extra copies of a gene (amplification; for example MET, HER2) or loss.
- Fusions: Accidental joining of two genes (for example ALK, ROS1, RET, NTRK, FGFR2). RNA-based testing may be preferred because some fusions can be missed on DNA panels.
- Genomic signatures: Microsatellite instability (MSI) and tumor mutational burden (TMB), calculated from the tumor's overall mutation pattern rather than a single gene and used for immunotherapy decisions.
Where Is NGS Used in Oncology?
1. Targeted Treatment Selection
NGS's most important oncology application is identifying tumor somatic alterations to find suitable targeted medicines. The table below summarizes major alterations sought in advanced disease. New approvals expand this list annually.
| Cancer | Principal alterations sought by NGS |
|---|---|
| Non-small cell lung | EGFR, ALK, ROS1, BRAF V600E, KRAS G12C, MET exon 14, RET, HER2 mutation, NTRK, NRG1 |
| Breast | PIK3CA, AKT1, PTEN, ESR1 (often in blood), inherited BRCA1/2 |
| Colorectal | KRAS/NRAS, BRAF V600E, MSI, HER2 amplification |
| Prostate | BRCA1/2 and other DNA repair (HRR) genes, MSI |
| Ovarian | BRCA1/2, homologous recombination deficiency (HRD) |
| Biliary tract (cholangiocarcinoma) | FGFR2 fusion, IDH1, HER2, BRAF V600E, MSI |
| Thyroid | RET, BRAF, NTRK |
We have discussed drugs developed for each alteration in separate articles: ALK for alectinib, HER2 for mutant lung cancer, T-DXd, PIK3CA for PI3K inhibitors, ESR1 for camizestrant and FGFR2 for lirafugratinib.
2. Tumor-Agnostic (Organ-Agnostic) Treatments
Some drugs have been approved for all tumors carrying a particular biomarker, regardless of the organ of origin. NGS is the most practical way to detect most such biomarkers, making comprehensive NGS particularly important in rare tumors and patients who have exhausted standard treatments.
| Biomarker | Drug | FDA approval |
|---|---|---|
| MSI-H / dMMR | Pembrolizumab | 23 May 2017 (first tumor-agnostic approval) |
| NTRK fusion | Larotrectinib; entrectinib; repotrectinib | November 2018; August 2019; June 2024 |
| TMB-H (≥10 mutations/megabase) | Pembrolizumab | 16 June 2020 (with FoundationOne CDx) |
| BRAF V600E | Dabrafenib + trametinib | June 2022 |
| RET fusion | Selpercatinib | September 2022 |
| HER2 (IHC 3+)* | Trastuzumab deruxtecan | April 2024 |
*HER2 protein overexpression is determined by immunohistochemistry (IHC), not NGS; it is included as complementary information. Dostarlimab is also approved for dMMR tumors.
3. Detecting Inherited Cancer Predisposition
NGS panels using blood or saliva examine inherited cancer genes such as BRCA1/2, Lynch syndrome genes (MLH1, MSH2, MSH6, PMS2), TP53, PALB2 in one test. This information affects both the patient's treatment, such as PARP inhibitors, and family screening and preventive measures. For details, consult preventive surgery in BRCA carriers in our related article.
An important point: Tumor tissue NGS can also suggest an inherited alteration. In a Memorial Sloan Kettering analysis of approximately 12.000 patients, %17 had a pathogenic or likely pathogenic inherited alteration and %8 had a clinically actionable alteration; many did not meet existing genetic testing criteria. Therefore, potentially inherited findings in a tumor report should be confirmed in blood and accompanied by genetic counseling.
4. Liquid Biopsy
Tumor cells release small DNA fragments into blood, circulating tumor DNA (ctDNA). NGS examination of these fragments helps select treatment when tissue cannot be obtained or is insufficient, and monitors resistance mutations arising during treatment, such as in breast cancer ESR1. Uses for detecting microscopic disease remaining after surgery, or minimal residual disease, remain largely investigational; we discussed the GALAXY study in ctDNA and colorectal cancer and blood tests for cancer screening in healthy people in Galleri in our related articles.
Liquid biopsy has two major pitfalls: a negative result may arise because the tumor sheds insufficient DNA, so a “negative” does not rule out tissue testing. Also, age-related mutations accumulating in blood cells unrelated to cancer (clonal hematopoiesis) may be mistakenly attributed to the tumor.
5. Other Uses
- Pharmacogenetics: Examining genes determining how the body processes certain medicines. For example, alterations in DPYD increase serious side-effect risk from chemotherapy such as 5-FU and capecitabine.
- Diagnosis and classification: Molecular alterations have become part of diagnosis, particularly in sarcomas, brain tumors and leukemias/lymphomas.
- Cancers of unknown primary: ESMO's 2024 recommendations advise NGS for these patients.
- Reproductive medicine: Genetic diagnosis before embryo transfer in couples carrying inherited cancer genes: preimplantation genetic diagnosis.
What Do Guidelines Say?
ESMO's Precision Medicine Working Group recommended routine NGS in 2020 for advanced nonsquamous non-small cell lung, prostate, ovarian and biliary tract cancers ; in colorectal cancer, NGS was accepted as an alternative to single-gene tests. The 2024 update added advanced breast cancer, certain rare cancers, including gastrointestinal stromal tumors, soft tissue sarcomas and thyroid cancer, and cancers of unknown primary to the list. Tumor-agnostic biomarkers (NTRK, RET, BRAF V600E, MSI-H, TMB-H) matter regardless of tumor type. United States NCCN guidelines also recommend “broad molecular profiling” in many advanced cancers.
Figure 3 · How to Read an NGS Report
Not every alteration in a report means the same thing. The finding type determines the next step.
Limitations and Realistic Expectations
NGS is powerful but does not guarantee a “target” for every patient. Among the first 10.336 patients tested with Memorial Sloan Kettering's MSK-IMPACT, approximately %37 of tumors had at least one actionable alteration, but approximately %11 of patients could enter trials based on these findings. Türkiye shows a similar picture: a single-center study presented at the 2026 Turkish Medical Oncology Congress examined 91 patients undergoing somatic NGS; %38 of findings had high-level evidence (ESCAT I-II), but %15 of patients received targeted treatment.
- The proportion of actionable alterations varies greatly by cancer type. It is high in lung adenocarcinoma and low in some cancers. Expecting a drug for every patient is unrealistic.
- There may be a gap between finding and treatment. A matching drug may not be licensed or reimbursed in our country, and clinical trial access may be limited.
- Variants of uncertain significance (VUS) are common and may cause anxiety; they are not used for treatment or family screening decisions.
- Tissue problems: Small biopsies and old or poorly preserved samples may cause test failure. Planning adequate tissue for NGS at diagnosis is important.
- Time: Results may take days to weeks. If treatment must begin rapidly, the oncologist should help plan which tests come first.
- Inherited findings: Tumor tests may reveal unexpected inherited findings; prior patient information and genetic counseling are important.
- “More genes are always better” is not true. What matters is reliable examination of the genes required for that cancer and interpretation by an experienced team.
The Situation in Türkiye
- SGK reimbursement: The fee schedule updated by the 21 April 2024 amendment to the Healthcare Implementation Communiqué (SUT) lists next-generation DNA sequencing procedures according to the number of genes examined (1 gene, 2-4, 5-15, 16-40 and 41 or more genes) under separate codes. Separate codes cover general NGS panels, somatic tumor mutation analysis and somatic mutation panels from blood cell-free DNA, or liquid biopsy. No specific cancer types are listed; billing frequency and per-gene restrictions are defined.
- Liquid biopsy requirement: Blood-based somatic mutation panels require a medical board report including at least one medical geneticist and one medical oncologist.
- Regulations: Genetic diagnostic centers are subject to the 2020 Regulation on Genetic Disease Evaluation Centers; the center director must specialize in medical genetics or pediatric genetics.
- Use is expanding: A study of 1.023 metastatic lung cancer patients from seven centers (2018-2025) found that %52 underwent NGS; use in 2025 exceeded %90. A targetable alteration was found in %28 of patients (EGFR %16, ALK %5).
- Private testing fees: These vary by institution and test; reliable current prices could not be obtained.
- Türkiye Genome Project: Announced in 2019 by the Ministry of Health and TÜSEB, the project aimed to sequence 100.000 people's genomes within a few years; verified information on its current status could not be obtained.
We track access to targeted medicines in our country in New FDA-Approved Cancer Drugs: Türkiye Access Map in our related article.
Frequently Asked Questions
- Should Every Cancer Patient Have NGS?
- No. Guidelines recommend NGS particularly for advanced lung, prostate, ovarian, biliary tract and breast cancers, cancers of unknown primary and certain rare tumors. Treatment decisions in many early cancers do not depend on NGS. Your oncologist decides whether it is needed.
- Is a New Biopsy Needed?
- Usually not; tissue collected at diagnosis and stored in pathology as a paraffin block can be used. If insufficient, a new biopsy or blood-based liquid biopsy may be considered.
- How Long Until Results?
- It varies by laboratory and test, generally from a few days to a few weeks.
- Are Tumor NGS and Inherited Genetic Testing the Same?
- No. Tumor testing seeks changes acquired later in tumor cells. Inherited testing uses blood or saliva to seek changes present from birth. Tumor testing may suggest an inherited alteration, but blood confirmation is required.
- My Report Says “Variant of Uncertain Significance.” Should I Worry?
- Generally not. It means whether the change is harmful is not yet known. These variants do not guide treatment, and most eventually prove harmless.
- Does Liquid Biopsy Replace Tissue Biopsy?
- Not entirely. Liquid biopsy is valuable when tissue cannot be obtained or for monitoring resistance mutations. However, if the tumor releases insufficient DNA into blood, false negatives may occur and tissue testing is needed.
- What If My NGS Finds No Target?
- This does not mean no treatment options exist. Chemotherapy, immunotherapy and other standard treatments remain available; new tests and drugs may also become relevant during the disease course.
Glossary
- Next-generation sequencing (NGS)
- Sequencing technology reading millions of DNA or RNA fragments simultaneously. Also called massively parallel sequencing.
- Sanger sequencing
- The conventional method developed in 1977, reading DNA as individual fragments.
- Read depth (coverage)
- How many times a DNA region is read. Greater depth improves reliability.
- Variant allele frequency (VAF)
- The percentage of reads covering a region that contain an alteration.
- Somatic / germline
- Somatic: a non-inherited alteration found only in the tumor. Germline: an inheritable alteration present from birth in all cells.
- Fusion gene
- An abnormal gene formed by joining two different genes; a target for many targeted treatments.
- MSI / TMB
- Microsatellite instability and tumor mutational burden: genomic signatures indicating overall tumor mutation levels and associated with immunotherapy response.
- ctDNA
- Cell-free tumor-derived DNA fragments circulating in blood.
- Variant of uncertain significance (VUS)
- A genetic alteration whose relationship to disease is not yet known.
- Companion diagnostic
- A test approved with a particular drug to determine which patients should receive it.
DROZDOGAN Academy Commentary
Over the past ten years, NGS has added “which genetic alteration does it carry?” alongside “which organ's cancer?” Today, selecting the best treatment without NGS is impossible in many cancers, particularly advanced lung cancer.
⚡ Why it matters
Examining dozens or hundreds of genes in one test uses tissue efficiently, saves time and avoids missing rare but treatable alterations, such asNTRK, RET, NRG1 fusions. Tumor-agnostic approvals have also made NGS indispensable in rare tumors.
✅ Strengths
High sensitivity; simultaneous mutation, fusion, copy-number and genomic signature (MSI, TMB) analysis; blood-based testing; detection of inherited predispositions. Its clinical role is established through FDA-approved companion diagnostics and ESMO recommendations. It is reimbursed by SGK in Türkiye, and lung cancer use is increasing rapidly.
⚠️ Limitations
Only some patients have actionable alterations, and only some obtain matching drugs. Results need experienced interpretation; uncertain variants and unexpected inherited findings may confuse patients. Tissue quality, time and cost are major practical barriers.
🩺 Practical steps
- If diagnosed with advanced cancer, ask your oncologist whether NGS is necessary for you as part of care.
- When planning biopsy, emphasize obtaining enough tissue for molecular tests as an important consideration.
- Always assess the report with your oncologist; if possible, request discussion at a molecular tumor board meeting.
- If the report suggests a potentially inherited alteration, seek genetic counseling ; it may also matter for your relatives.
- Regarding “cancer gene tests” sold online without physician referral, exercise caution.
❓ Open questions
Will whole-genome sequencing become routine tumor testing? Will blood-based residual disease tests change postoperative treatment decisions? How much will artificial intelligence accelerate NGS interpretation? Most importantly, how can access to matching treatments for all identified targets be ensured?
Our Brief Verdict: Next-generation sequencing is a fundamental diagnostic tool in modern oncology. It is a guideline-recommended standard in advanced lung, prostate, ovarian, biliary tract and breast cancers and cancers of unknown primary. However, NGS is not a “miracle test”; its value depends on performing it in the right patient at the right time and an experienced team translating findings into treatment.
- This article updates and expands our earlier NGS article. It was prepared from publicly available sources as of September 2026: the United States National Human Genome Research Institute, Ensembl, Nobel Foundation, Illumina, FDA, CMS, Foundation Medicine technical documents, corporate press releases, professional news sources, the SGK communiqué, Official Gazette and studies published from Türkiye.
- Corrections to the previous version: Humans have not 23 but 23 pairs (46) of chromosomes; protein-coding gene numbers are approximately not 25.000 but 19.900; the protein-coding portion is approximately %1,5 of the genome; before 2010, Sanger sequencing (1977) and commercial NGS (after 2005) were already used; NGS can also read the entire genome; tumor tests use hundreds of reads per region, not 10-20.
- Information from secondary sources: The scope of ESMO 2024 recommendations comes from a company summary because the primary paper was inaccessible; tumor-tissue inherited alteration rates (%17 and %8) from a professional journal news report; and some tumor-agnostic approval dates from professional news. An encyclopedic source was used for early commercial NGS platform history.
- SUT information: Codes and conditions come from a summary of the communiqué dated 21 April 2024; consult SGK's operative text for current implementation. Private test fees and the Türkiye Genome Project's current status could not be verified.
- Based on general knowledge: The table of alterations sought by cancer type, clonal hematopoiesis, DPYD pharmacogenetics, the advantage of RNA testing for fusions and NCCN recommendations are presented as established oncology knowledge.
- The “Our Assessment” section and Academy commentary are our own.
- Diagrams derive from verified statements in the text and contain no new data.
- This article is not personal medical advice. Genetic testing decisions should be made with your oncologist and, when needed, a medical geneticist.
References
- National Human Genome Research Institute. Human Genome Project fact sheet; The cost of sequencing a human genome; Genetics glossary: Exome, Chromosome.
- NHGRI. Researchers generate the first complete, gapless sequence of a human genome. 31 March 2022.
- Ensembl 116 (GENCODE 50, GRCh38.p14). Human genome annotation statistics. June 2026.
- Nobel Prize Outreach. Frederick Sanger, Nobel Prize in Chemistry 1980: facts.
- Illumina. An introduction to next-generation sequencing; Sequencing technology; Sequencing coverage.
- Illumina. NovaSeq X Series launch. Press release, 29 September 2022.
- Foundation Medicine. FoundationOne CDx and FoundationOne Liquid CDx technical information labels.
- FDA / Thermo Fisher. FDA approves first companion diagnostic test to simultaneously screen for multiple non-small cell lung cancer therapies (Oncomine Dx Target Test). 22 June 2017.
- Memorial Sloan Kettering. FDA authorizes MSK-IMPACT test for analyzing patient tumors. 15 November 2017; NCI Cancer Currents: genomic profiling tests.
- CMS. FDA announces approval, CMS proposes coverage of first breakthrough-designated test to detect extensive number of cancer biomarkers (FoundationOne CDx). 30 November 2017.
- Guardant Health. FDA approves first liquid biopsy next-generation sequencing companion diagnostic test. 7 August 2020.
- OncLive. FDA approves FoundationOne Liquid CDx for solid tumors. August 2020; Tumor-agnostic approvals of T-DXd and repotrectinib carry the field forward. 2024.
- FDA. FDA grants accelerated approval to pembrolizumab for first tissue/site agnostic indication. 23 May 2017.
- FDA. FDA approves larotrectinib for solid tumors with NTRK gene fusions. 26 November 2018.
- FDA. FDA approves pembrolizumab for adults and children with TMB-H solid tumors. 16 June 2020.
- Mosele MF et al. Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024: a report from the ESMO Precision Medicine Working Group. Ann Oncol. 2024 (via the SOPHiA Genetics summary).
- CAP TODAY. Room to grow: tumor-germline sequencing (Stadler et al., JCO 2021).
- Social Security Institution. Communiqué Amending the Healthcare Implementation Communiqué, ANNEX-2/B. Official Gazette, 21 April 2024, Issue 32524.
- Regulation on Genetic Disease Evaluation Centers. Official Gazette, 10 January 2020, Issue 31004.
- Karaoğlu et al. NGS use in metastatic non-small cell lung cancer: multicenter Türkiye data. Genes. 2025;16(12):1446.
- Yazkaç et al. Somatic NGS results and access to targeted treatment. Turkish Medical Oncology Congress 2026 abstract.
- Yeni Şafak. Mapping the genetics of Turkish people (Türkiye Genome Project). 5 November 2019.
Sources
Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification.
Int J Mol Sci, Feb 2017.
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