Monday, June 24, 2013

Open access comparative analysis of NIPD methods

Here is a new paper comparing several approaches in non-invasive prenatal diagnostics (NIPD) published in CCLM in June and is available for free at:

It was submitted almost a year ago, but the data is still current.


Rapidly developing next-generation sequencing (NGS) technologies produce a large amount of data across the whole human genome and allow a large number of DNA samples to be analyzed simultaneously. Screening cell-free fetal DNA (cffDNA) obtained from maternal blood using NGS technologies has provided new opportunities for non-invasive prenatal diagnosis (NIPD) of fetal aneuploidies. One of the major challenges to the analysis of fetal abnormalities is the development of accurate and reliable algorithms capable of analyzing large numbers of short sequence reads. Several such algorithms have recently been developed. Here, we provide a review of recent NGS-based NIPD methods as well as the available algorithms for short-read sequence analysis. We furthermore introduce the practical application of these algorithms for the detection of different types of fetal aneuploidies, and compare the performance, cost and complexity of each approach for clinical deployment. Our review identifies several main technologies and trends in NGS-based NIPD. The main considerations for clinical development for NIPD and screening tests using DNA sequencing are: accuracy, intellectual property, cost and the ability to screen for a wide range of chromosomal abnormalities and genetic defects. The cost of the diagnostic test depends on the sequencing method, diagnostic algorithm and volume of the tests. If the cost of sequencing equipment and reagents remains at or around current levels, targeted approaches for sequencing-based aneuploidy testing and SNP-based methods are preferred.


Since the discovery of the cell-free fetal nucleic acid sequences in maternal peripheral blood, several methods for highly accurate and highly sensitive aneuploidy testing using NGS technology either for full genome sequencing or sequencing of targeted areas of the genome were developed. Prenatal tests utilizing these methods are already offered as screening tests for trisomy 21, 18 and 13, reducing the need for risky invasive procedures. Additional clinical trials are underway to validate these methods for use as diagnostic tests for both high-risk pregnancies and screening of the general population. The final decision on the implementation of a NGS-based test for NIPD of aneuploidy in clinical practice should be based on the criteria of high diagnostic accuracy, clinical and cost-effectiveness and the ability to make a diagnosis even in cases where the content of cffDNA is low. Furthermore, large-scale validation studies should be carried out independent from the tests’ manufacturing companies. Tests implemented in a clinical setting should not be time consuming, which is very important in prenatal diagnosis. It is also important to take into account the nationality of the patients in order to implement the test in clinics around the world. Tests should also require a minimal cost of equipment and infrastructure in order to be available to small laboratories around the world. Today NGS-based tests for diagnosis of trisomies 21, 18 and 13 may be combined with ultrasonographic detection and serum markers for more accurate diagnosis of fetal aneuploidy, in order to avoid invasive procedures. Methods utilizing full genome sequencing allow for accurate detection of other autosomal and sex aneuploidies, but are limited by the high cost of sequencing. Sequencing of targeted areas of the genome allows one to significantly lower the cost of sequencing while providing high accuracy and sensitivity in diagnosing common aneuploidies. Methods utilizing parental genotypes, where DNA from one or both parents is available, in addition to common trisomy detection, provide for highly accurate counts of autosomes and sex chromosomes and can be performed using significantly cheaper and easier to operate sequencing equipment. Our review demonstrated that NGS-based NIPD is a rapidly evolving field with many research teams developing and commercializing tests using new technologies and performing large scale clinical trials. As the new NGS technologies become available, new methods for NIPD will be developed that allow the analysis of a broader spectrum of chromosomal abnormalities and genetic diseases, and cost will be reduced. Several commercial NIPD providers developed proprietary fetal quantifiers and protocols for increasing diagnostic accuracy of the tests and these may not be publicly available. All of the reviewed methods bear equipment, technology, cost, intellectual property and performance risk; thus, careful consideration should be given to each of these aspects when deploying or developing NGS-based NIPD in a clinical setting.

Sequencing requirements (see table 2 in the paper for Verinata, Ariosa and Sequenom):

Nepomnyashchaya, Y. N., Artemov, A. V., Roumiantsev, S. A., Roumyantsev, A. G., & Zhavoronkov, A. (2012). Non-invasive prenatal diagnostics of aneuploidy using next-generation DNA sequencing technologies, and clinical considerations.