Performance of a Paired-End Sequencing-Based Noninvasive Prenatal Screening Test in the Detection of Genome-Wide Fetal Chromosomal Anomalies.

Background: Noninvasive prenatal tests (NIPTs) detect fetal chromosomal anomalies with high clinical sensitivity and specificity. We examined the performance of a paired-end sequencing-based NIPT in the detection of genome-wide fetal chromosomal anomalies including common trisomies, sex chromosomal aneuploidies (SCA), rare autosomal aneuploidies (RAAs), and partial deletions/duplications ≥7 Mb.

Methods: Frozen plasma samples from pregnant women were tested using the VeriSeq NIPT Solution v2 assay.

Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing.

Objective: Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction.

Method: The MaterniT21™ PLUS test uses massively parallel sequencing to analyze cell-free fetal DNA in maternal plasma and detect chromosomal abnormalities. We assess the impact of a variety of factors, both maternal and fetal, on the fetal fraction across a large number of samples processed by Sequenom Laboratories.

Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts.

Objective: This study introduces a novel method, referred to as SeqFF, for estimating the fetal DNA fraction in the plasma of pregnant women and to infer the underlying mechanism that allows for such statistical modeling.

Methods: Autosomal regional read counts from whole-genome massively parallel single-end sequencing of circulating cell-free DNA (ccfDNA) from the plasma of 25 312 pregnant women were used to train a multivariate model. The pretrained model was then applied to 505 pregnant samples to assess the performance of SeqFF against known methodologies for fetal DNA fraction calculations.

Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains.

Background: Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.

Detection of fetal subchromosomal abnormalities by sequencing circulating cell-free DNA from maternal plasma.

Background: The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect.

Non-invasive prenatal chromosomal aneuploidy testing--clinical experience: 100,000 clinical samples.

Objective: As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries.

Noninvasive detection of a balanced fetal translocation from maternal plasma.

Background: Massively parallel sequencing of circulating cell free (ccf) DNA from maternal plasma has been demonstrated to be a powerful method for the detection of fetal copy number variations (CNVs). Although the detection of CNVs has been described by multiple independent groups, genomic aberrations resulting in copy number-neutral events including balanced translocations have proven to be more challenging to detect noninvasively from ccf DNA.

Methods: Data modeling was initially performed to evaluate multiple methods, ultimately leveraging the short length of ccf DNA and paired-end sequencing to construct read-specific mapping characteristics.

Noninvasive prenatal screening for fetal trisomies 21, 18, 13 and the common sex chromosome aneuploidies from maternal blood using massively parallel genomic sequencing of DNA.

Objective: The objective of this study was to validate the clinical performance of massively parallel genomic sequencing of cell-free deoxyribonucleic acid contained in specimens from pregnant women at high risk for fetal aneuploidy to test fetuses for trisomies 21, 18, and 13; fetal sex; and the common sex chromosome aneuploidies (45, X; 47, XXX; 47, XXY; 47, XYY).

Study Design: This was a prospective multicenter observational study of pregnant women at high risk for fetal aneuploidy who had made the decision to pursue invasive testing for prenatal diagnosis. Massively parallel single-read multiplexed sequencing of cell-free deoxyribonucleic acid was performed in maternal blood for aneuploidy detection.

Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies.

Purpose: We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies.

Methods: Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

Objective: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes.

Method: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype.

High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.

Background: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations.

Objective: Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies.

Method: Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies (17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid triplet pregnancies [Correction made here after initial online publication.

Detection of microdeletion 22q11.2 in a fetus by next-generation sequencing of maternal plasma.

Background: Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

Purpose: To determine whether maternal plasma cell-free DNA sequencing can effectively identify trisomy 18 and 13.

Methods: Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.

DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.

Purpose: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement.

Methods: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome.

Clinical validation of a genetic model to estimate the risk of developing choroidal neovascular age-related macular degeneration.

Predictive tests for estimating the risk of developing late-stage neovascular age-related macular degeneration (AMD) are subject to unique challenges. AMD prevalence increases with age, clinical phenotypes are heterogeneous and control collections are prone to high false-negative rates, as many control subjects are likely to develop disease with advancing age. Risk prediction tests have been presented previously, using up to ten genetic markers and a range of self-reported non-genetic variables such as body mass index (BMI) and smoking history.

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

Objective: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA.

Study Design: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed.

A genomic analysis of the archaeal system Ignicoccus hospitalis-Nanoarchaeum equitans.

Background: The relationship between the hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans is the only known example of a specific association between two species of Archaea. Little is known about the mechanisms that enable this relationship.

Results: We sequenced the complete genome of I.

On the predictability of protein database search complexity and its relevance to optimization of distributed searches.

We discuss several aspects related to load balancing of database search jobs in a distributed computing environment, such as Linux cluster. Load balancing is a technique for making the most of multiple computational resources, which is particularly relevant in environments in which the usage of such resources is very high. The particular case of the Sequest program is considered here, but the general methodology should apply to any similar database search program.

Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae.

The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology. The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci.

Proteomic survey of metabolic pathways in rice.

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues.

Analysis of quantitative proteomic data generated via multidimensional protein identification technology.

We describe the analysis of quantitative proteomic samples via multidimensional protein identification technology (MudPIT). Ratio amounts of the soluble portion of the S. cerevisiae proteome from cultures of S.