[Explained by a Physician] The NIPT Testing Method Using Next-Generation DNA Sequencing

Revised: August 21, 2025

A physician explains the mechanism, accuracy, and comparison with other technologies of NIPT using NGS. We cover the effects of gestational age and BMI, and the importance of confirmatory diagnosis, based on evidence.

Introduction

When considering prenatal testing, the "accuracy" and "peace of mind" that a test offers are extremely important points. In recent years, "Non-Invasive Prenatal Testing (NIPT)," which can estimate the risk of fetal chromosomal abnormalities from a simple maternal blood draw, has attracted significant attention.

In particular, NIPT using Next-Generation Sequencing (NGS) offers higher accuracy and comprehensiveness than conventional technologies, and has become the standard method worldwide. NIPT's clinical application began in earnest around 2011, and in the roughly fifteen years since, it has spread rapidly, with tens of millions of tests performed across more than 90 countries. [ref:3]

Compared with conventional maternal serum marker tests such as the triple marker test or quad test, NIPT achieves a far higher detection rate and lower false-positive rate. While maternal serum marker tests detected Down syndrome at a rate of roughly 80-85%, NGS-based NIPT has reported a sensitivity of over 99%, illustrating just how far testing technology has advanced. [ref:1]

Another major advantage is that, because NIPT only requires a maternal blood draw, it carries none of the miscarriage risk associated with conventional amniocentesis or chorionic villus sampling, placing very little physical burden on the pregnant woman. Amniocentesis and chorionic villus sampling are associated with a miscarriage risk of roughly 0.1-0.3%, whereas NIPT carries no such risk. This combination of non-invasiveness and high accuracy is arguably the biggest reason NIPT has become so widespread around the world.

In Japan too, the system for providing NIPT has expanded year by year. NIPT began as clinical research in 2013, and in 2022 a framework for certified facilities was established under Ministry of Health, Labour and Welfare guidelines, creating an environment that is increasingly accessible to more pregnant women. [ref:4] That said, testing is still being conducted in parallel at non-certified facilities, and the need for more robust genetic counseling systems before and after testing has been pointed out as a challenge. [ref:5]

This article provides an easy-to-understand explanation of how NGS works, how it differs from other technologies, and points to keep in mind when undergoing testing. We hope this offers useful information both for those who are unsure whether to be tested and for those who want to know more about the technology involved. [ref:1]

How NGS Works: The Technology for Highly Accurate Reading of Fetal DNA

NGS (Next-Generation Sequencing) is a technology that analyzes vast numbers of DNA fragments all at once. Whereas the conventional Sanger sequencing method could only read one strand of DNA at a time, NGS can read millions to billions of DNA fragments simultaneously. This "Massively Parallel Sequencing (MPS)" is NGS's greatest innovation, and it is the foundational technology that made NIPT possible.

In NIPT, cfDNA (cell-free DNA) of fetal origin contained in the mother's blood is collected, and this is examined for signs of fetal chromosomal abnormalities. cfDNA refers to short DNA fragments released into the blood when cells naturally break down (apoptosis). cfDNA fragments are extremely short, only about 150-200 base pairs long, a length that corresponds to roughly one nucleosome's worth of DNA. [ref:3]

With NGS, the cfDNA is first amplified, and each fragment (read) is read in detail one by one. A computer then compares each piece of DNA to determine which human chromosome it belongs to. By statistically analyzing the amount of DNA derived from each chromosome, the test can estimate, for example, that an excess of chromosome 21 indicates a higher likelihood of trisomy 21 (Down syndrome).

This process incorporates error correction and algorithmic statistical analysis, enabling highly accurate detection even from tiny amounts of DNA. Specifically, a statistical measure called a z-score is used to numerically evaluate whether the number of reads assigned to each chromosome deviates significantly from the normal range. Compared with conventional tests, NGS's major strength lies in its ability to detect signs of abnormality more non-invasively and comprehensively.

cfDNA is released into the mother's blood mainly when placental chorionic villus cells break down, and it reflects the fetus's genetic information. Because the concentration of cfDNA rises as pregnancy progresses, knowing the appropriate timing for testing is also important. Generally, at 10 weeks of pregnancy the proportion of fetal-derived cfDNA (the "fetal fraction") is around 10-15%, and this proportion rises further as pregnancy advances. Ensuring a sufficient fetal fraction allows for more reliable analysis results. [ref:1]

A Detailed Look at the NIPT Analysis Process Using NGS

NIPT analysis using NGS goes through several important steps. Understanding this process is helpful for appreciating the reliability of the test.

1. Blood collection and plasma separation: About 10-20 mL of blood is drawn from the pregnant woman, and plasma is separated to extract cfDNA. Dedicated collection tubes (cfDNA-preservation tubes) are often used, which prevent cfDNA degradation even at room temperature. After collection, the sample is promptly centrifuged to remove blood cell components and recover the plasma.
2. Library preparation: Specific tags (adapters) are attached to the extracted cfDNA, processing it into a form readable by NGS. This step makes it possible to identify which sample each DNA fragment came from. A technique called "multiplexing," which processes multiple samples simultaneously, improves cost efficiency while still accurately distinguishing individual samples.
3. Sequencing (reading the base sequence): An NGS device reads the base sequences of millions to tens of millions of DNA fragments all at once. Current mainstream platforms can generate hundreds of gigabytes to terabytes of data in a single run, and ensuring sufficient read depth (sequencing depth) improves detection accuracy.
4. Mapping and counting: Each DNA fragment that has been read is compared against the human reference genome to determine which chromosome it came from. The number of reads assigned to each chromosome is then counted. This process incorporates multiple quality-control steps, including correction for GC-content bias and duplicate reads.
5. Statistical analysis and judgment: Statistical algorithms are used to evaluate whether the read count for each chromosome shows an abnormal deviation from the expected value. For example, if the read count for chromosome 21 is statistically significantly elevated, trisomy 21 is suspected. The final risk assessment is made comprehensively, taking the fetal fraction value into account as well.

In this way, NGS-based NIPT achieves high detection accuracy by going through multiple quality-control steps. By combining large-scale computer data processing with advanced statistical analysis, it is possible to derive reliable results even from minute amounts of cfDNA. NIPT analysis approaches broadly fall into two categories: "whole genome sequencing (WGS)" and "targeted sequencing." WGS reads the entire genome uniformly and is suited for comprehensively screening all chromosomes for numerical abnormalities. Targeted sequencing, on the other hand, selectively reads specific chromosomal regions at high depth, allowing for highly sensitive detection even with a smaller amount of data.

NGS, Microarray, and PCR: A Comparison of Prenatal Testing Technologies

There are several technologies used in prenatal testing, each with different characteristics and applications. The representative methods are NGS (next-generation sequencing), microarray analysis, and real-time PCR. [ref:2]

Real-time PCR is a technique that amplifies and measures a narrow set of specific genes or chromosomal regions. It has the advantages of fast detection and low cost, but since it cannot detect abnormalities outside its target, its testing scope is limited. It is generally used to check for specific known mutations or the presence of infectious agents, and it is not well suited for comprehensively examining all chromosomes. PCR works by repeating cycles of raising and lowering temperature to denature, anneal, and extend the DNA double strand, exponentially amplifying the target DNA region. Real-time PCR uses fluorescent probes to monitor the amount of amplification in real time, yielding quantitative results.

Microarray analysis uses a DNA chip that can examine hundreds of thousands of chromosomal regions or more, making it particularly strong at detecting structural abnormalities such as microdeletions and microduplications. Microarray chips are densely arrayed with short DNA probes corresponding to known genomic regions, and the hybridization (complementary binding) pattern between the sample DNA and the probes is detected as a fluorescent signal. However, because its sensitivity is lower than that of NGS, it is not well suited to minute samples such as cfDNA. Microarray analysis is often used as a confirmatory diagnostic method, applied to cells obtained through amniocentesis or chorionic villus sampling.

By contrast, NGS can comprehensively analyze all chromosomes even from minute amounts of DNA such as cfDNA, making it the technology best suited for non-invasively estimating fetal chromosomal numerical abnormalities from maternal blood. NGS allows detection sensitivity to be flexibly controlled by adjusting the read depth (sequencing depth), and thanks to technological advances, analysis costs have been falling year after year. In fact, the running cost of NGS has fallen to less than one-thousandth of what it was a decade ago, greatly accelerating its implementation in clinical settings. [ref:2]

Summary of the Advantages and Disadvantages of Each Technology

The characteristics of the three main prenatal testing technologies can be summarized as follows.

• NGS (next-generation sequencing): Can comprehensively analyze all chromosomes even from minute amounts of cfDNA. Best suited for NIPT. High statistical precision and a low false-positive rate. However, analysis can sometimes take several days to about a week. In recent years, automation and speed improvements have steadily shortened turnaround times.
• Microarray analysis: Can detect minute chromosomal deletions and duplications at high resolution. Particularly strong for analyzing fetal cells obtained directly through confirmatory diagnosis. Faces sensitivity challenges with minute samples such as cfDNA. On the other hand, it remains a useful technology for efficiently screening for known pathogenic variants.
• Real-time PCR: Enables fast, low-cost detection focused on specific targets. However, it cannot detect chromosomal abnormalities outside its target, and lacks comprehensiveness as a screening tool. It remains widely used clinically today for specific purposes, such as infectious disease testing and Rh blood type determination.

As shown above, each technology has its own strengths and weaknesses, and the appropriate method is chosen depending on the purpose of the test and the type of sample. For NIPT, which targets minute amounts of cfDNA in maternal blood, NGS is adopted worldwide as the most suitable technology.

Does Gestational Age or Physical Constitution Affect Results? The Accuracy of NGS and the Importance of Confirmatory Diagnosis

NGS-based NIPT is highly accurate, but test results can be affected by the pregnant woman's physical condition and gestational age. Understanding these factors in advance can help you interpret test results more appropriately.

First, testing is only possible from 10 weeks of pregnancy onward. This is because cfDNA in maternal blood reaches a sufficient concentration around 10 weeks. Fetal-derived cfDNA is thought to make up roughly 10-20% of the total cfDNA in maternal blood, but this proportion is low in early pregnancy, making it difficult to obtain sufficient data. At many testing institutions, a fetal fraction below 4% is classified as "indeterminate," requiring a retest or additional measures. Testing before this point may result in an "indeterminate" outcome.

In addition, when the mother's BMI is high, the proportion of fetal-derived cfDNA relative to maternal-derived DNA (the "fetal fraction") may decrease, potentially lowering detection sensitivity. This occurs because, in women with a higher BMI, more cfDNA is released from maternal fat cells and other sources, relatively diluting the fetal-derived cfDNA. Research has reported that in pregnant women with a BMI over 30, the fetal fraction tends to be significantly lower. [ref:3] Twin pregnancies and irregular menstrual cycles are other factors that can affect the reliability of test results.

Even more importantly, NIPT is, after all, only a screening test. Because it analyzes fetal DNA circulating in maternal blood, even a high-risk result does not necessarily mean the fetus definitely has the condition in question. This is because cfDNA is mainly derived from the placenta, and "confined placental mosaicism (CPM)," a condition in which the placenta and fetus have different chromosomal makeups, can be a cause of false positives.

For this reason, when NIPT returns a high-risk result, a confirmatory diagnostic test such as amniocentesis or chorionic villus sampling is necessary. Because confirmatory amniocentesis and chorionic villus sampling examine fetal cells directly, they can accurately diagnose the presence or absence of chromosomal abnormalities. Amniocentesis is typically performed around 15-18 weeks of pregnancy, and chorionic villus sampling around 11-14 weeks.

It is important for pregnant women themselves to understand that, while NGS-based NIPT is a powerful screening test, it is not infallible, and only becomes a fully adequate basis for decision-making when combined with confirmatory diagnosis.

Why NIPT Accuracy Is Not 100%

NIPT is a screening test with a very high detection rate, but it is not a confirmatory diagnosis. Understanding the main reasons its accuracy does not reach 100% is important for correctly interpreting test results.

• Confined placental mosaicism (CPM): In cases where the placenta and fetus have different chromosomal makeups, NIPT can show false positives or false negatives. Because cfDNA is mainly placenta-derived, it does not always directly reflect the fetus's own chromosomes. CPM occurs at a frequency of roughly 1-2%, making it one of the major causes of false positives in NIPT.
• Insufficient fetal fraction: If the proportion of fetal-derived cfDNA in maternal blood is low, it becomes difficult to accurately detect numerical chromosomal abnormalities. This is more likely to occur when testing is done too early in pregnancy or when the mother's BMI is high. When the fetal fraction is below 3-4%, the reliability of the test drops significantly, and many facilities recommend a repeat blood draw.
• Maternal factors: It has been reported that the mother's own chromosomal mosaicism, or, rarely, the presence of a maternal malignant tumor, can affect the cfDNA analysis results. Because tumor cells also release cfDNA (ctDNA: circulating tumor DNA), unexpected chromosomal abnormality patterns can appear, and cases have been reported in the literature in which maternal cancer was incidentally discovered through NIPT.
• Multiple pregnancies: In pregnancies with twins or more, differences in the cfDNA ratio contributed by each fetus can make accurate assessment more difficult. In particular, because the interpretation of the fetal fraction differs between monochorionic and dichorionic twin pregnancies, results require more careful interpretation.
• Technical limitations: NGS read accuracy and bioinformatics analysis algorithms also have their limits, and not all minute abnormalities can be detected. In particular, low-level mosaicism and structural chromosomal translocations are considered difficult to detect with current NIPT.

For these reasons, even when NIPT returns a "high-risk" result, rather than jumping to a definitive conclusion, it is recommended to always consult a specialist and undergo confirmatory diagnosis. Conversely, a "low-risk" result does not guarantee a 100% absence of abnormality, so it remains important to continue with regular prenatal checkups. NIPT's positive predictive value (PPV) varies considerably depending on the prevalence (prior probability) of the condition in question. For example, for trisomy 21, the positive predictive value can exceed 80% in pregnant women aged 35 or older, but may drop to around 50% in younger pregnant women, making it essential to make a comprehensive judgment that takes age and other risk factors into account. [ref:3]

The History of NIPT and Its Global Adoption

The history of NIPT traces back to 1997, when Professor Yuk Ming Dennis Lo of the Chinese University of Hong Kong discovered the presence of fetal-derived cell-free DNA in maternal plasma. [ref:3] This groundbreaking discovery was the first to demonstrate the possibility of non-invasively obtaining fetal genetic information, and it fundamentally changed the course of prenatal testing thereafter.

In the 2000s, as NGS technology costs fell and processing speed improved, screening for chromosomal abnormalities using cfDNA began to be considered a realistic clinical application. In 2008, a group led by Professor Stephen Quake at Stanford University in the United States demonstrated that fetal trisomy 21 could be detected by analyzing cfDNA in maternal plasma using NGS, opening the way for NIPT's clinical implementation.

Between 2011 and 2012, commercial NIPT testing services began primarily in the United States and China. Initially, testing focused mainly on the three major trisomies-trisomy 21, trisomy 18, and trisomy 13-but as the technology advanced, sex chromosome abnormalities and microdeletion syndromes were also added to the scope of testing.

In Japan, NIPT began as clinical research in April 2013, under guidelines from the Japan Society of Obstetrics and Gynecology. Initially it was offered only at a limited number of certified facilities, but as awareness and demand for the test rapidly grew, a certification system introduced by the Japanese Association of Medical Sciences in 2022 helped advance the development of the testing infrastructure. [ref:4] Today, tens of thousands of tests are performed annually in Japan, and NIPT is becoming an established option among prenatal tests.

Globally, NIPT is widely adopted in many countries, including the United States, European nations, China, and Australia, and in some countries it is covered by public health insurance. For example, in European countries such as the Netherlands and Belgium, NIPT is offered as the first-line screening test for all pregnant women. [ref:5]

The Role and Importance of Genetic Counseling

Undergoing genetic counseling both before and after NIPT is strongly recommended. Genetic counseling is a process in which a physician with expert knowledge of genetic medicine, or a certified genetic counselor, explains the significance of the test, its risks, and how to interpret the results, in an easy-to-understand way, to the pregnant woman and her partner, supporting their autonomous decision-making.

• Pre-test counseling: Explanations are given about the characteristics of the conditions targeted by NIPT, the accuracy and limitations of the test (the possibility of false positives and false negatives), and the difference between screening and confirmatory diagnosis. This includes support for making a self-determined decision about whether to undergo the test, based on sufficient information.
• Post-test counseling: Especially when a high-risk result is returned, the counselor helps ensure the meaning of the result is correctly understood, and presents concrete options for next steps (such as undergoing confirmatory diagnosis or referral to a specialist). Comprehensive support, including psychological support, is provided.
• For low-risk results: Because a low-risk result does not definitively mean "no abnormality," explanations are given about the correct interpretation of the result and the continued importance of regular prenatal checkups.

In Japan, certified facilities operating under the Japanese Association of Medical Sciences' certification system are required to provide genetic counseling both before and after testing. On the other hand, it has been reported that some facilities offering NIPT outside the certification system do not have adequate counseling systems in place. [ref:5] Because NIPT results carry very significant meaning for pregnant women and their families, receiving the test under appropriate counseling is just as important a factor as the accuracy of the test itself.

Making an Evidence-Based, Reassuring Choice

NGS-based NIPT is a powerful screening test that can non-invasively evaluate fetal chromosomal abnormalities at an early stage. It is technically highly accurate and offers many advantages compared with other methods.

At the same time, it is important to correctly understand its limitations related to gestational age and maternal conditions, as well as the situations in which confirmatory diagnosis becomes necessary. NIPT is, after all, only part of a screening process, and it is important to calmly determine the next steps based on the results.

Above all, how one responds after receiving NIPT results is the most important point for the pregnant woman and her family. Even if a high-risk result is returned, it is important to first consult a specialist physician or counselor and gather sufficient information about the need for confirmatory diagnosis and the options available afterward. Choosing tests based on scientific evidence and receiving appropriate medical support will bring peace of mind to both the individual and her family.

Prenatal testing technology continues to evolve day by day, and we are entering an era in which more accurate and safer tests are available. As NGS technology continues to innovate, next-next-generation technologies such as long-read sequencing and single-molecule sequencing are expected to enable the detection of even finer abnormalities and the use of new biomarkers. We encourage you to gain accurate knowledge from a trusted specialist institution and make the choice that is best for you.

Test 8 Genetic Disease Risks at Japan's Lowest Price with the "Non-Invasive Prenatal Testing (NIPT)"

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Frequently Asked Questions

Q1. From how many weeks can I take NGS-based NIPT?

A. NIPT is recommended from 10 weeks of pregnancy onward. This is because fetal-derived cfDNA (cell-free DNA) in maternal blood reaches a concentration sufficient for testing around 10 weeks. Before that point, the fetal fraction may be insufficient, resulting in an "indeterminate" outcome. Many testing institutions recommend a retest when the fetal fraction is below 4%.

Q2. If NIPT shows "high risk," does that mean the fetus definitely has an abnormality?

A. No. NIPT is only a screening test, and a high-risk result simply indicates a "higher likelihood." False positives can occur due to factors such as confined placental mosaicism (CPM), so anyone who receives a high-risk result needs to undergo confirmatory diagnosis via amniocentesis or chorionic villus sampling. The positive predictive value also varies depending on maternal age and disease prevalence.

Q3. Does a high BMI affect the accuracy of NIPT?

A. Yes. When the mother's BMI is high, maternal-derived cfDNA increases, which can relatively lower the proportion of fetal-derived cfDNA (the fetal fraction) and reduce detection sensitivity, according to reports. This effect tends to be more pronounced with a BMI of 30 or above, but cases where testing becomes impossible are limited, so we recommend first consulting a specialized medical institution.

Q4. What is the difference between NGS, microarray analysis, and PCR?

A. NGS can comprehensively analyze all chromosomes from a minute amount of cfDNA, making it the technology best suited for NIPT. Microarray analysis is strong at detecting minute chromosomal deletions and duplications, but faces sensitivity challenges with minute samples such as cfDNA. Real-time PCR excels at rapid detection of specific targets, but cannot comprehensively examine all chromosomes. [ref:2]

Q5. How long does it take to get NIPT results?

A. Generally, results are available about 1-2 weeks after the blood draw. NGS-based analysis requires a certain amount of time because it involves sequencing an enormous number of DNA fragments and performing statistical processing. The turnaround time varies by testing institution, so please check with the institution you use for details.

Q6. What kinds of chromosomal abnormalities can NIPT check for?

A. Standard NIPT targets the three major trisomies: trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). Depending on the testing institution, additional items such as sex chromosome abnormalities, other numerical chromosomal abnormalities, and certain microdeletion syndromes may also be available.

Q7. Can NIPT take the place of confirmatory diagnosis?

A. No. NIPT cannot take the place of confirmatory diagnosis. NIPT is a screening test that assesses the level of risk. A final diagnosis requires a confirmatory test that directly examines fetal cells, such as amniocentesis or chorionic villus sampling. It is important to decide on next steps based on the NIPT results.

Q8. Is genetic counseling necessary before undergoing NIPT?

A. Yes. Undergoing genetic counseling both before and after NIPT is strongly recommended. Going into the test with sufficient information about its significance, its limitations, and how to respond to the results allows you to interpret the outcome more appropriately. At facilities certified by the Japanese Association of Medical Sciences, pre- and post-test counseling is a requirement.

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Author

M.D., Ph.D.
Tasuku Hiroshige

Ph.D. in Medicine; Board-Certified Specialist and Instructor, Japanese Urological Association; Certified Physician, Japanese Society of Medical Oncology; Specialist, Japanese Society of Anti-Aging Medicine; Certified Occupational Physician, Japan Medical Association; Certified Physician, Japanese Society of Chemotherapy; Certified Physician, Japanese Society for Sexually Transmitted Infections; Certificate of da Vinci System Training As a Console Surgeon, and others
After graduating from Kagoshima University School of Medicine in 2010, he built extensive clinical experience as a urologist. In addition to his clinical work, he is also actively engaged in academic activities, including conference presentations, writing papers, and securing research funding. He holds specialist qualifications across a wide range of fields, including urology, cancer treatment, anti-aging medicine, and infectious disease treatment. Drawing on his extensive medical knowledge and skills, he provides care tailored to each individual patient.

[References]

(1) J Biol Chem, March 1997
(2) seeDNA Genetic Testing & DNA Testing, July 2025
(3) Part 1: Prenatal Diagnosis - The Governance of Non-Invasive Prenatal Testing (NIPT) in Japan: Up to the Start of Clinical Research - Ritsumeikan University Center for Ars Vivendi
(4) The Yomiuri Shimbun
(5) JST "Health and Medical Transformation," March 2023