Existing NIPTs can be classified in two categories based on the genomic material that is analysed: whole genome and chromosome specific (targeted).
Whole genome analysis was the first-generation technology of NIPT that was developed in 2011. These tests use massively parallel shotgun sequencing (MPSS) to randomly sequence the cell free DNA (cfDNA) extracted from a pregnant woman’s blood. There is no way to control which fragments are sequenced and analysed using this method. Therefore, they suffer from the following disadvantages:
Low Read Depth: Whole genome based NIPTs only read a small fraction of the human genome as a trade-off for sequencing time and cost. The number of times a nucleotide in the genome is read during sequencing is called the read-depth. These methods have a read-depth of 0.1X to 0.5X. This lowers the statistical accuracy of these NIPTs.
Copy Number Variants (CNVs): Some parts of the human genome may be present in more or less than two copies. These regions are known as copy number variants. Whole-genome based NIPTs cannot distinguish between changes due to aneuploidies and copy number variants. This is a major source of false results in whole-genome based NIPTs, according to recent publications.
Non-linear Amplification: Because these tests cannot exclude certain regions from analysis, they are prone to higher error arising from polymerase chain reaction (PCR). Genomic regions rich in G and C nucleotides, and regions with complex genomic architecture are difficult to amplify and sequence because of their PCR bias. These regions are underrepresented and the bias needs to be corrected during analysis. This also lowers the statistical accuracy of these NIPTs.
Inaccurate or No Fetal Fraction Measurement: Fetal fraction is the measure of the proportion of circulating cfDNA of fetal origin. Due to low read-depth, whole genome based NIPTs cannot discriminate between cfDNA of maternal and fetal origin. So, they either do not report fetal fraction or use crude global regression models to estimate the fetal fraction. The probability of a false negative is very high below 4% fetal fraction and accuracy at this threshold is very critical.
Chromosome Specific (Targeted) Analysis
Chromosome specific or targeted analysis was the second-generation technology for NIPT that was developed in 2013. Amplicon analysis has the following disadvantages: Some of them are chromosome specific but they differ significantly in their analysis method. The other one uses haplotype reconstruction of the fetal genome for aneuploidy detection whereas other uses microarrays as a counting method for aneuploidy detection. Some of them use PCR amplification of target regions for their chromosome specific analysis.
Deduplication is not possible: Amplicons are clones of the cfDNA fragments that are created using PCR. Amplicons have the same size; therefore, it is not possible to remove duplicates. PCR Error Propagation: If an error happens during the initial PCR steps, this error is propagated during the next steps. This makes it sometimes difficult to distinguish differences in the genomic region due to SNPs from those due to PCR errors. No access to original fragments: Because the amplicons are all similar, information such as fragment length that can be used to discriminate between maternal and fetal cfDNA cannot be extracted.
Other than these general disadvantages due to amplicons, Panorama and Harmony have the following specific disadvantages based on their analysis methods: 1: One of them relies on reconstructing the fetal haplotype by sequencing cfDNA from the plasma that contains both maternal and fetal material, and then removing from it the maternal haplotype obtained by sequencing the buffy coat. Millions of models are then fit to this reconstructed haplotype to determine any potential aneuploidy. This method has a high redraw rate due to low fetal fraction, and has lower accuracy in case of consanguineous pregnancies. 2: One of them uses microarrays to distinguish the difference in genetic material from the different chromosomes to detect any potential aneuploidy. Because microarrays are analog methods that depend on differences in fluorescence intensities for measurement, they have lower sensitivity and specificity than NIPTs that count using sequencing.
VERACITY Target Capture Technology
NIPD Genetics has developed a proprietary technology for non-invasive prenatal diagnosis of chromosomal abnormalities and potential mutations located in any region of the human genome. This technology is being currently marketed internationally as the VERACITY non-invasive prenatal test for the detection of fetal aneuploidies of chromosomes 21, 18, 13, X and Y with the option of also determining fetal sex.
Our novel technology captures and counts cfDNA fragments from selected genomic regions using targeted enrichment and next generation sequencing (NGS) with proprietary genetic and analytical tools. VERACITY considers the complexities of the human genome, and is designed to avoid problematic regions that reduce the sensitivity and specificity of other NIPTs. It is based on established, robust genetic technologies such as DNA hybridization, enrichment, and sequencing, and it is simple to implement. VERACITY directly analyzes maternal and fetal DNA fragments unlike amplicon based NIPTs. Therefore, it has access to additional information which is used to enrich the assay, remove duplicate data and improve the accuracy of the test. The main features and advantages of VERACITY are:
Targeted Genomic Analysis: VERACITY avoids regions with copy number variants that have been shown to reduce the sensitivity and specificity of whole-genome based NIPTs. The regions are also chosen to have similar GC content to minimize the effect of GC bias during amplification and sequencing. Regions with complex genomic architecture that prevent linear amplification are also avoided. Thus, VERACITY has been designed to have very high sensitivity and specificity in detecting aneuploidies.
High Read-Depth: Because VERACITY analyses a small portion of the human genome, it does not compromise read-depth. VERACITY has a median read-depth of 500X which is orders of magnitude greater than that achievable by whole-genome based NIPTs. High read-depth increases the statistical confidence in the results.
High Level of Multiplexing: VERACITY can also analyse more samples per sequencing run than whole genome based NIPTs because the sequencing reads span selected targeted regions and are not wasted on random, irrelevant regions of the genome. The high level of multiplexing reduces the cost of the test making it more accessible.
Accurate Fetal Fraction Measurement: VERACITY uses informative loci to differentiate between maternal and fetal cfDNA fragments. It then uses the high read-depth of these loci to calculate the minor allele frequency and measure the fetal fraction. The method is extremely accurate, even in lower fetal fraction cases ensuring the accuracy of VERACITY in detecting aneuploidies and avoiding false negative results.
Microdeletions, Microduplications and Point Mutations: VERACITY can detect changes at the sub-chromosomal level. The technology can be used to create additional assays for large and small microdeletions and microduplications, and potentially point mutations. Whole-genome based NIPTs do not have sufficient read-depth to detect these changes accurately, and therefore have very poor sensitivity and specificity. VERACITY’s targeted, high-read depth approach to detection ensures the high sensitivity and specificity of these additional NIPT panels.
VERACITY, uses specifically designed and accredited internationally
accredited target capture sequences(TACS) in order to avoid CNVs, repetitive elementss and complex genomic
structures. This target specific approach increases sensitivity, reliability and accuracy of VERACITY by ensuring to overcome the issues other NIPTs have
Read-depth is the number of times a nucelotide in the genome is read during analysis. VERACITY captures DNA fragments only from targeted regions on chromosomes of interest. So, it is able to read these selected regions at an extremely high read-depth which improves the statistical accuracy of the analysis and increases the sensitivity and specificity of VERACITY.
Fetal Fraciton Measurement
VERACITY uses informative loci to reliably distinguish fetal cfDNA from maternal cfDNA. A proprietary bioinformatics software uses the high read depth counts of these informative loci to accurately calculate the fetal fraction. Accurate fetal fraction measurements raise the robustness and reliability of VERACITY.