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Persian gene raazman | NGS
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Today, there are five different NGS approaches to gaining access to genetic information, the most common of which is the technology provided by Illumina Company and use of its completely exclusive method, Sequencing by Synthesis (SBS). In this technology, after creating a DNA library from the studied source, sequencing is done, in which the members of each library are formed by linking the linker molecules to the two ends of the linear DNA molecules. The library’s DNA source can be genome DNA, RNA molecules, or specific DNA sequences that have been increased by PCR.

In this method, proliferation in order to sequencing is on flow cell and using Bridge-PCR. After preparing and placing it on flow cell and carrying out Bridge-PCR, collections of target molecules with a density of about 1 million double-strand molecules are created, which, by denaturing them, provides a single strand pattern for sequencing by fluorescent nucleotides. In this method, each nucleotide is blocked by a fluorophore in the 3’OH position, which after the nucleotide is placed in the growing strand, the emitted signal is recorded, and then a new phase of synthesis begins. By continuing this method, the nucleotide sequences of the desired molecules are prepared for the user with high accuracy. Today, with the help of this technology, sequencing different sources from a nucleic acid are possible with high accuracy and low cost.

Some of the NGS capabilities using SBS method:

  1. Whole Genome Sequencing
  2. Whole Exome Sequencing
  3. Transcriptome Sequencing
  4. Methylome Sequencing
  5. Small RNA Sequencing
  6. ChIP Sequencing
  7. De Novo Sequencing
  8. Metagenomics Sequencing

NIPT Testing

Non-Invasive Prenatal Testing (NIPT)

In 1997, scientists discovered the presence of embryonic DNA in pregnant women’s blood. This important discovery, which was carried out by tracing parts of the embryo Y chromosome in the mother’s serum by Dennis Lo, was fundamental to the emergence of non-invasive methods for diagnosis of aneuploidy during pregnancy. The principles of this method are based on the measurement of free DNA in cell-free DNA, which is released from placental cells. The researchers consider this discovery to be a revolution in Perinatology science and predict that in the near future, tests based on it would replace the common screening methods. Finally, true success was achieved in 2008, when two independent groups of researchers with a study on two populations of 18 and 14 of the embryos suffering from aneuploidy, proved the ability to detect embryonic trisomy using Massively Parallel Sequencing technique with an accuracy of more than 99%.

The non-invasive molecular technique of the detection of aneuploidy diagnosis

Generally, the focus of screening programs in prenatal period is on Down syndrome diagnosis, NTDs (1), and two other autosomal aneuploidy including trisomy 18 (Edema’s syndrome) and trisomy 13 (Patau syndrome), which are less common. Down syndrome or trisomy 21 is the most common aneuploidy in humans with a prevalence of approximately 1 in 800. The risk of developing an embryo into Down syndrome increases with age, with a 45-year-old woman at risk of 1 to 35. Today, medical science advances have made it possible to treat most patients with this syndrome, so that people with Down syndrome can live up to the ages (about 55). Trisomy 18 and 13, like Down syndrome, are more common in the first and second trimester than in the childbirth time, and mostly spontaneous abortion occurs in them. Patients with trisomy 18 and 13 have a short life span, and only 5 to 10% of them survive until one year of age.

Microdeletion Syndromes

Deletion syndromes are considered as a group of clinically diagnostic disorders that are caused by the removal of part of the chromosome. The size and location of the deletion, determine the characteristics of clinical manifestations and their severity. Clinical manifestations can include growth delays, mental disabilities, growth differences, behavioral problems, nutritional difficulties, low muscle strength, seizure and dysmorphic.

Sexual chromosome aneuploidies

Sexual chromosome aneuploidies include numerical abnormalities in chromosomes X and Y, either by deletion or addition. Although the clinical symptoms of the patients are generally slight and without mental disabilities, some have a phenotypic problem, such as physical dysfunction, delay in learning, and sterility.

In addition to embryonic sex, in this test, Sexual chromosome aneuploidies like Turner syndrome are also recognized from the tenth week.

Over the past two decades, various methods including measure chemical markers and ultrasound parameters, as well as the combination of both of them have been developed for screening Down syndrome, but scientists have always been looking for better ways. The biggest problem in traditional screening methods is the high percentage of false positive and negative results, and in the confirmatory tests is the abortion probability due to being invasive of the sampling method. In the NIPT method, without the need for cells of placenta tissue or sampling from amniotic fluid (amniocentesis), a few millilitres of maternal venous blood are used, and the percentage of false positive and negative cases is reduced by less than 1%.

NIPT sequencing millions of maternal and fetal DNA fragments in each sample. Using the Next Generation Sequencing technology of the entire genome and four different bioinformatics analyzes, the information throughout the entire genome is analyzed and compared with standard references to identify genetic disorders.

The longevity of this DNA is very short and less than 2 hours and immediately disappears from the mother’s blood after childbirth, so the previous pregnancy will not cause the disorder as a result of the test. To prevent a decrease in the amount of DNA, the mother’s blood is taken in tubes containing a specific substance, which maintains the DNA content unchanged. More populous studies have recently been reported and indicated that the detection rate of this method for trisomy 21 is 99.1% and with a specificity of 99.1 to 99.9%. In order to get more familiar with the statistics discussed,   if we assume that the total pregnancy in Iran is one million three hundred thousand per year, then the following benefits can be achieved if screening is carried out using the new NIPT method:

  1. In the common method, the number of patients with false-positive results that are introduced for the amniocentesis confirmatory testing is about 65,000 patient, but in the NIPT method, this number has reached to the lowest possible rate.
  2. The number of abortion cases due to referral for invasive sampling is approximately 650 in the first method and in the new method only 6 cases.
  3. False negative results in the traditional screening method are approximately 325 cases, but in the new method will be 1 or 2 cases, in other words, 323 fewer patients in one year.

It should be noted that implementing this test did not eliminate the need for (AFP) testing for examination of NTDs in the second trimester, and the pregnant woman should necessarily refer to the lab in the 15th to 20th week of pregnancy.

It should be noted that for individuals who have positive result (high risk) about cell-free DNA testing, a confirmatory test such as CVS or amniocentesis should be carried out, but given the very low false-positive percentage, the number of cases requiring this will be very slight.

Test Benefits: being non-invasive, only a few ml of the mother’s blood is needed to do the test. High sensitivity and accuracy, studies on large populations show feature and sensitivity of more than 99%. This test can be done from the tenth week of pregnancy and this time helps to make better decisions. Although by 2013, much research has been done on the usefulness of this test for high-risk pregnancies, but in 2014, two highly acclaimed centers -the American College of Medical Genetics and the Royal College of Obstetrics and Gynecology- as well as Professor Kypros Nicolaides have suggested the use of NIPT as a high-sensitivity screening test.


* As a second-stage test, for those who are considered as high-risk individuals.

  1. Pregnant women whose screening test results are positive.
  2. Women older than 35 years old
  3. Pregnant women who have been diagnosed high-risk in sonography examinations.
  4. Those with a history of a previous pregnancy with chromosomal abnormalities.
  5. A person who has been pregnant with IVF or who already has had repeated abortions.
  6. Individuals that have prohibition in the use of invasive tests such as Placenta Parvia, those who are at high risk of abortion and those with hepatitis B infection.

* As a screening test with a very high detection capability, for those who do not want to accept the false negative risk rate in the common screening methods.

* A selection as a confirmation test for women who are not able to accept the risk of invasive methods, or are afraid of the way of sampling in invasive techniques.

* An option for cases where cell cultures fail.


  1. pregnant women should be consulted prior to making a decision about choosing the method used for screening or confirming the positive result (high risk) in common screening methods, and to advise on the benefits and disadvantages of all available methods.
  2. The application of this test is for those who are afraid of how the sampling of invasive methods.
  3. NIPT testing for women who can not accept abortion risk in invasive methods, such as women who have been pregnant after long and costly treatments or at old ages.