Arnold Oliphant

Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18.

OBJECTIVEnWe sought to develop a novel biochemical assay and algorithm for the prenatal evaluation of risk for fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA obtained from maternal blood.nnnSTUDY DESIGNnWe assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies. We used digital analysis of selected regions in combination with a novel algorithm, fetal-fraction optimized risk of trisomy evaluation (FORTE), to determine trisomy risk for each subject.nnnRESULTSnIn all, 163/171 subjects in the training set passed quality control criteria. Using a Z statistic, 35/35 T21 cases and 7/7 T18 cases had Z statistic >3 and 120/121 disomic cases had Z statistic <3. FORTE produced an individualized trisomy risk score for each subject, and correctly discriminated all T21 and T18 cases from disomic cases. All 167 subjects in the blinded validation set passed quality control and FORTE performance matched that observed in the training set correctly discriminating 36/36 T21 cases and 8/8 T18 cases from 123/123 disomic cases.nnnCONCLUSIONnDigital analysis of selected regions and FORTE enable accurate, scalable noninvasive fetal aneuploidy detection.

Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy.

To develop a novel prenatal assay based on selective analysis of cell‐free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).

Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma

To determine the effects of gestational age and maternal weight on percent fetal cell‐free DNA (cfDNA) in maternal plasma and the change in fetal cfDNA amounts within the same patient over time.

Whole‐Genome Genotyping

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecans GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecans capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.

Trisomy 13 detection in the first trimester of pregnancy using a chromosome‐selective cell‐free DNA analysis method

To assess the performance of chromosome‐selective sequencing of maternal plasma cell‐free DNA (cfDNA) in non‐invasive prenatal testing for trisomy 13.

Description of the data from the Collaborative Study on the Genetics of Alcoholism (COGA) and single-nucleotide polymorphism genotyping for Genetic Analysis Workshop 14

The data provided to the Genetic Analysis Workshop 14 (GAW 14) was the result of a collaboration among several different groups, catalyzed by Elizabeth Pugh from The Center for Inherited Disease Research (CIDR) and the organizers of GAW 14, Jean MacCluer and Laura Almasy. The DNA, phenotypic characterization, and microsatellite genomic survey were provided by the Collaborative Study on the Genetics of Alcoholism (COGA), a nine-site national collaboration funded by the National Institute of Alcohol and Alcoholism (NIAAA) and the National Institute of Drug Abuse (NIDA) with the overarching goal of identifying and characterizing genes that affect the susceptibility to develop alcohol dependence and related phenotypes. CIDR, Affymetrix, and Illumina provided single-nucleotide polymorphism genotyping of a large subset of the COGA subjects. This article briefly describes the dataset that was provided.

A highly informative SNP linkage panel for human genetic studies

We have developed a highly informative set of single-nucleotide polymorphism (SNP) assays designed for linkage mapping of the human genome. These assays were developed on a robust multiplexed assay system to provide a combination of very high accuracy and data completeness with high throughput for linkage studies. The linkage panel is comprised of approximately 4,700 SNPs with 0.39 average minor allele frequency and 624-kb average spacing. Based on almost 2 million genotypes, data quality was shown to be extremely high, with a 99.94% call rate, >99.99% reproducibility and 99.995% genotypes consistent with mendelian inheritance. We constructed a genetic map with an average 1.5-cM resolution using series of 28 CEPH pedigrees. The relative information content of this panel was higher than those of commonly used STR marker panels. The potent combination of this SNP linkage panel with the multiplexed assay system provides a previously unattainable level of performance for linkage studies.

The use of random-sequence oligonucleotides for determining consensus sequences

Publisher Summary This chapter discusses the utilization of random-sequence oligonucleotides to determine consensus sequences. It describes the development of an alternative method that is useful in defining the sequence requirements of a genetic element. A collection of recombinant deoxyribonucleic acid (DNA) molecules is made in which random or highly degenerate DNA replaces a genetic element of interest. A selection or screen is made to isolate from this collection those sequences that confer an equivalent function. A comparison is made of DNA sequences that satisfy a particular selection results in a consensus that defines the genetic element. Thus, unlike conventional mutagenesis, which uncovers the nonfunctional derivatives of a wild-type sequence, the method presented in the chapter uses the bias of selective pressure to select functional sequences from random DNA. In principle, the method is applicable for any genetic element that confers a phenotype that is subject to a selection or screen. The first step in determining the consensus sequence for such a genetic element is to construct an appropriate vector. The crucial features of the vector are as follows: (1) it lacks the genetic element of interest, (2) it contains all other sequences necessary to pass the selection, and (3) it contains unique restriction endonuclease cleavage sites that can be used to clone DNA segments at the position of the deleted genetic element. Oligomers were synthesized and kindly provided by Alexander Nussbaum using the phosphite triester method on an Applied Biosystems DNA Synthesizer Model 380A. The three variations of this method are described in the chapter and have important implications concerning the design of further experiments on other genetic elements.

Microarray-Based Cell-Free DNA Analysis Improves Noninvasive Prenatal Testing

Objective: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. Methods: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. Results: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. Conclusions: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.

Non‐invasive risk assessment of fetal sex chromosome aneuploidy through directed analysis and incorporation of fetal fraction

To assess the performance of a directed chromosomal analysis approach in the prenatal evaluation of fetal sex chromosome aneuploidy.