Optimal Detection of Fetal Chromosomal Abnormalities by Massively Parallel DNA Sequencing of Cell-Free Fetal DNA from Maternal Blood

Verinata Health, Inc., San Carlos, CA, USA.
Clinical Chemistry (Impact Factor: 7.91). 04/2011; 57(7):1042-9. DOI: 10.1373/clinchem.2011.165910
Source: PubMed


Massively parallel DNA sequencing of cell-free fetal DNA from maternal blood can detect fetal chromosomal abnormalities. Although existing algorithms focus on the detection of fetal trisomy 21 (T21), these same algorithms have difficulty detecting trisomy 18 (T18).
Blood samples were collected from 1014 patients at 13 US clinic locations before they underwent an invasive prenatal procedure. All samples were processed to plasma, and the DNA extracted from 119 samples underwent massively parallel DNA sequencing. Fifty-three sequenced samples came from women with an abnormal fetal karyotype. To minimize the intra- and interrun sequencing variation, we developed an optimized algorithm by using normalized chromosome values (NCVs) from the sequencing data on a training set of 71 samples with 26 abnormal karyotypes. The classification process was then evaluated on an independent test set of 48 samples with 27 abnormal karyotypes.
Mapped sites for chromosomes of interest in the sequencing data from the training set were normalized individually by calculating the ratio of the number of sites on the specified chromosome to the number of sites observed on an optimized normalizing chromosome (or chromosome set). Threshold values for trisomy or sex chromosome classification were then established for all chromosomes of interest, and a classification schema was defined. Sequencing of the independent test set led to 100% correct classification of T21 (13 of 13) and T18 (8 of 8) samples. Other chromosomal abnormalities were also identified.
Massively parallel sequencing is capable of detecting multiple fetal chromosomal abnormalities from maternal plasma when an optimized algorithm is used.

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Available from: Amy Sehnert, May 24, 2014
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    • "Notably, the important guanine and cytosine nucleotide contents of chromosomes 18 and 13, may result in insensitive detection for those chromosomes.7,24,26 Some optimized technical protocols, to correct this guanine and cytosine bias,37–39 produced a high detection rate for trisomy 18,18,27–29,38 but due to the lower incidence of trisomy 13, the number of cases available for evaluation by genomic technologies and gNIPT has been smaller. Ten to 25 recruited cases were reported in four studies,18,27,29,38 while the number of nontrisomy cases in the same studies analyzed ranged from 264–1,939. "
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    ABSTRACT: Current prenatal diagnosis for fetal aneuploidies (including trisomy 21 [T21]) generally relies on an initial biochemical serum-based noninvasive prenatal testing (NIPT) after which women who are deemed to be at high risk are offered an invasive confirmatory test (amniocentesis or chorionic villi sampling for a fetal karyotype), which is associated with a risk of fetal miscarriage. Recently, genomics-based NIPT (gNIPT) was proposed for the analysis of fetal genomic DNA circulating in maternal blood. The diffusion of this technology in routine prenatal care could be a major breakthrough in prenatal diagnosis, since initial research studies suggest that this novel approach could be very effective and could reduce substantially the number of invasive procedures. However, the limitations of gNIPT may be underappreciated. In this review, we examine currently published literature on gNIPT to highlight advantages and limitations. At this time, the performance of gNIPT is relatively well-documented only in high-risk pregnancies for T21 and trisomy 18. This additional screening test may be an option for women classified as high-risk of aneuploidy who wish to avoid invasive diagnostic tests, but it is crucial that providers carefully counsel patients about the test's advantages and limitations. The gNIPT is currently not recommended as a first-tier prenatal screening test for T21. Since gNIPT is not considered as a diagnostic test, a positive gNIPT result should always be confirmed by an invasive test, such as amniocentesis or chorionic villus sampling. Validation studies are needed to optimally introduce this technology into the existing routine workflow of prenatal care.
    Full-text · Article · Jul 2014 · The Application of Clinical Genetics
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    • "The choice of sequencing parameters needs to be balanced against costs. Previous studies have shown that an acceptable level of test sensitivity can be reached with relatively short read lengths (36–52 bp) and modest coverage (2–10 million reads per sample) (Liang et al., 2013; Sehnert et al., 2011). "
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    ABSTRACT: Non-invasive prenatal testing (NIPT) of fetal aneuploidy using cell-free fetal DNA is becoming part of routine clinical practice. RAPIDR (Reliable Accurate Prenatal non-Invasive Diagnosis R package) is an easy-to-use open-source R package that implements several published NIPT analysis methods. The input to RAPIDR is a set of sequence alignment files in the BAM format, and the outputs are calls for aneuploidy, including trisomies 13, 18, 21 and monosomy X as well as fetal sex. RAPIDR has been extensively tested with a large sample set as part of the RAPID project in the UK. The package contains quality control steps to make it robust for use in the clinical setting.Availability and implementation: RAPIDR is implemented in R and can be freely downloaded via CRAN from here: information: Supplementary data are available at Bioinformatics online.
    Full-text · Article · Jul 2014 · Bioinformatics
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    • "Target-specific detection methods, including PCR (digital [12] or otherwise) and targeted sequencing, represent the most common current technologies for NIPT, while next-generation sequencing technology, which infers the entire fetal genome, is emerging as a feasible approach [13–15]. This enrichment in fetal DNA is valuable for these analytical platforms which detect and measure fetal aneuploidy in a background of normal maternal ploidy. "
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    ABSTRACT: Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.
    Preview · Article · Aug 2013 · PLoS ONE
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