Furthermore, the number of samples run together is an important consideration with this technology, as a high level of multiplexing can place a limitation on the total number of sequence reads carried out on each sample resulting in a reduced read depth, which in consequence can also affect accuracy in fetal fraction estimation. As such, the main limitations comprise (i) amplification variability leading to GC content and mappability bias that result in variable ratios of reads between chromosomes of interest, i.e., accuracy of fetal aneuploidy detection is not the same for all chromosomes and (ii) the need for pre-determined cut-offs based on past data, in an attempt to alleviate the aforementioned biases. Almost all MPSS based NIPT methods employ counting based (using read depth information) statistical methods to identify fetal chromosomal and sub-chromosomal copy number aberrations. the reads obtained from the reference chromosomes. The aneuploidy status of fetal chromosome of interest is determined by the number of sequence reads obtained from the chromosome of interest vs. This approach allows for tens of millions of short-sequence DNA fragments to be sequenced rapidly and simultaneously in a single run. Whole genome sequencing (WGS) was the first generation technology for NIPT that uses massively parallel shotgun sequencing (MPSS) to randomly sequence cfDNA extracted from a pregnant woman’s blood in a genome-wide manner. The most common commercially available techniques that utilize cfDNA analysis are described herein with their corresponding strengths and limitations ( Table 1). Such retrievable quantities of cfDNA thus led to the use as an important screening tool in early pregnancy. The average fetal fraction is 10–15% between 10 and 20 weeks of gestation. The fetal fraction represents the percentage of fetal cfDNA in relation to the overall circulating cell-free DNA in maternal plasma, and it is a major determinant of assay sensitivity. The discovery of cell-free fetal DNA (cffDNA) in the maternal circulation and the introduction of NGS has allowed the use and analysis of cffDNA in NIPT applications. In this review we compare the different technologies that provide the framework for NIPT, discuss implementation, recommendations from professional societies and highlight important considerations for genetic counselling. With further technical developments the screening is expanded to other genetic conditions such as sex chromosome anomalies (SCAs), rare autosomal trisomies (RATs), microdeletions/microduplications, structural chromosomal aberrations and monogenic diseases. A number of NIPT tests based on whole genome and targeted methods employing Next Generation Sequencing (NGS) have been developed and applied in clinical practice, ,. NIPT has been endorsed by professional bodies and organizations as a primary screening method regardless of the pregnancy risk status and is rapidly being adopted as a first choice for aneuploidy screening for trisomy 13, 18, 21. Furthermore, its accuracy is improved compared to other non-invasive screening approaches such as measurement of maternal serum biochemical markers combined with fetal ultrasound markers. NIPT is now widely adopted in the clinical setting as it provides no risk for the pregnancy compared to traditional invasive methods which entail a modest but significant risk of miscarriage of about 0.1–2%. NIPT analyses residual amounts of cfDNA that is circulating in the mother’s blood which consists of both maternal and fetal components. Non-invasive prenatal testing (NIPT) is a screening method for detecting potential fetal genetic abnormalities from cell-free DNA (cfDNA) in maternal circulation.
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