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. All samples were previously tested with a laboratory-developed NIPT and had known clinical outcomes. Individuals performing the sequencing were blinded to clinical outcome data. Clinical sensitivity and specificity were determined for basic (chromosomes 21, 18, 13, X, and Y) and genome-wide screening modes. RESULTS: Of 2335 samples that underwent genome-wide analysis, 28 did not meet QC requirements, resulting in a first-pass assay failure rate of 1.2%. Basic screening analysis, excluding known mosaics, correctly classified 130/130 trisomy 21 samples (sensitivity >99.9%, 95% confidence interval [CI] 97.1%-100%), 41/41 trisomy 18 samples (sensitivity >99.9%, 95% CI 91.4%-100%), and 26/26 trisomy 13 samples (sensitivity >99.9%, 95% CI 87.1%-100%) with 6 false-positive results; specificities ≥99.90% were reported for all 3 trisomies. Concordance for SCAs ranged from 90.5%-100%. Genome-wide screening analysis including known mosaics correctly classified 27/28 RAAs and 20/27 partial deletions/duplications with a specificity of 99.80% for both anomalies, and an overall genome-wide specificity for all anomalies of 99.34%. CONCLUSIONS: The VeriSeq NIPT Solution v2 assay enables accurate identification of fetal aneuploidy, allowing detection of genome-wide fetal chromosomal anomalies with high clinical sensitivities and specificities and a low assay failure rate.Clinical Trial Notification [CTN] identification number [ID]: CT-2018-CTN-01585-1 v1, Protocol: NIPT T05 002.

Lipid nanoparticle purification by spin centrifugation-dialysis (SCD): a facile and high-throughput approach for small scale preparation of siRNA-lipid complexes.

This paper describes the use of spin centrifugation-dialysis (SCD) for small-scale concentration/purification of siRNA-lipid complexes designed for use as therapeutic agents for gene silencing. SCD consists of a two-step method for concentration, filtration and buffer exchange of lipid nanoparticles (LNP) to provide a homogeneous preparation suitable for injection. Here, we compare SCD with the more traditionally used tangential flow filtration (TFF), and demonstrate the physicochemical and biological comparability of LNPs produced with both methods. TFF is a highly scalable method used in both developmental and production applications, but is limited in terms of miniaturization. In contrast to TFF, SCD is faster, less expensive, and requires less oversight for assembling LNPs for small-scale applications, such as target screening both in vitro and in vivo. The finding that SCD is a viable method for filtering LNPs in a manner similar to TFF, producing particles with comparable properties and biological activity, is significant given the complexity and sensitivity of LNPs to processing conditions.

Development of a microarray platform for FFPET profiling: application to the classification of human tumors.

BACKGROUND: mRNA profiling has become an important tool for developing and validating prognostic assays predictive of disease treatment response and outcome. Archives of annotated formalin-fixed paraffin-embedded tissues (FFPET) are available as a potential source for retrospective studies. Methods are needed to profile these FFPET samples that are linked to clinical outcomes to generate hypotheses that could lead to classifiers for clinical applications. METHODS: We developed a two-color microarray-based profiling platform by optimizing target amplification, experimental design, quality control, and microarray content and applied it to the profiling of FFPET samples. We profiled a set of 50 fresh frozen (FF) breast cancer samples and assigned class labels according to the signature and method by van 't Veer et al 1 and then profiled 50 matched FFPET samples to test how well the FFPET data predicted the class labels. We also compared the sorting power of classifiers derived from FFPET sample data with classifiers derived from data from matched FF samples. RESULTS: When a classifier developed with matched FF samples was applied to FFPET data to assign samples to either "good" or "poor" outcome class labels, the classifier was able to assign the FFPET samples to the correct class label with an average error rate = 12% to 16%, respectively, with an Odds Ratio = 36.4 to 60.4, respectively. A classifier derived from FFPET data was able to predict the class label in FFPET samples (leave-one-out cross validation) with an error rate of approximately 14% (p-value = 3.7 x 10(-7)). When applied to the matched FF samples, the FFPET-derived classifier was able to assign FF samples to the correct class labels with 96% accuracy. The single misclassification was attributed to poor sample quality, as measured by qPCR on total RNA, which emphasizes the need for sample quality control before profiling. CONCLUSION: We have optimized a platform for expression analyses and have shown that our profiling platform is able to accurately sort FFPET samples into class labels derived from FF classifiers. Furthermore, using this platform, a classifier derived from FFPET samples can reliably provide the same sorting power as a classifier derived from matched FF samples. We anticipate that these techniques could be used to generate hypotheses from archives of FFPET samples, and thus may lead to prognostic and predictive classifiers that could be used, for example, to segregate patients for clinical trial enrollment or to guide patient treatment.

Reagent preparation and storage for amplification of microarray hybridization targets with a fully automated system.

The advent of automated systems for gene expression profiling has accentuated the need for the development of convenient and cost-effective methods for reagent preparation. We have developed a method for the preparation and storage of pre-aliquoted cocktail plates that contain all reagents required for amplification of nucleic acid by reverse transcription and in vitro transcription reactions. Plates can be stored at -80 degrees C for at least 1 month and kept in a hotel at 4 degrees C for at least 24 h prior to use. Microarray data quality generated from these pre-aliquoted reagent plates is not statistically different between cRNA amplified with stored cocktails and cRNA amplified with freshly prepared cocktails. Deployment of pre-aliquoted, stored cocktail plates in a fully automated system not only increases the throughput of amplifying cRNA targets from thousands of RNA samples, but could also considerably reduce reagent costs and potentially improve process robustness.

Regulation of gene expression by lithium and depletion of inositol in slices of adult rat cortex.

Lithium inhibits inositol monophosphatase at therapeutically effective concentrations, and it has been hypothesized that depletion of brain inositol levels is an important chemical alteration for lithium's therapeutic efficacy in bipolar disorder. We have employed adult rat cortical slices as a model to investigate the gene regulatory consequences of inositol depletion effected by lithium using cytidine diphosphoryl-diacylglycerol as a functionally relevant biochemical marker to define treatment conditions. Genes coding for the neuropeptide hormone pituitary adenylate cyclase activating polypeptide (PACAP) and the enzyme that processes PACAP's precursor to the mature form, peptidylglycine alpha-amidating monooxygenase, were upregulated by inositol depletion. Previous work has shown that PACAP can increase tyrosine hydroxylase (TH) activity and dopamine release, and we found that the gene for GTP cyclohydrolase, which effectively regulates TH through synthesis of tetrahydrobiopterin, was also upregulated by inositol depletion. We propose that modulation of brain PACAP signaling might represent a new opportunity in the treatment of bipolar disorder.

A microarray platform comparison for neuroscience applications.

To address the need for high sensitivity in gene expression profiling of small neural tissue samples ( approximately 100 ng total RNA), we compared a novel RT-PCR-IVT protocol using fluor-reverse pairs on inkjet oligonucleotide microarrays and an RT-IVT protocol using 33P labeling on nylon cDNA arrays. The comparison protocol was designed to evaluate these systems for sensitivity, specificity, reproducibility, and linearity. We developed parameters, thresholds, and testing conditions that could be used to differentiate various systems that spanned detection chemistry and instrumentation; probe number and selection criteria; and sample processing protocols. We concluded that the inkjet system had better performance in sensitivity, specificity, and reproducibility than the nylon system, and similar performance in linearity. Between these two platforms, the data indicates that the inkjet system would perform better for the transcriptional profiling of 100 ng total RNA samples for neuroscience studies.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Microarray standard data set and figures of merit for comparing data processing methods and experiment designs.

MOTIVATION: There is a very large and growing level of effort toward improving the platforms, experiment designs, and data analysis methods for microarray expression profiling. Along with a growing richness in the approaches there is a growing confusion among most scientists as to how to make objective comparisons and choices between them for different applications. There is a need for a standard framework for the microarray community to compare and improve analytical and statistical methods. RESULTS: We report on a microarray data set comprising 204 in-situ synthesized oligonucleotide arrays, each hybridized with two-color cDNA samples derived from 20 different human tissues and cell lines. Design of the approximately 24 000 60mer oligonucleotides that report approximately 2500 known genes on the arrays, and design of the hybridization experiments, were carried out in a way that supports the performance assessment of alternative data processing approaches and of alternative experiment and array designs. We also propose standard figures of merit for success in detecting individual differential expression changes or expression levels, and for detecting similarities and differences in expression patterns across genes and experiments. We expect this data set and the proposed figures of merit will provide a standard framework for much of the microarray community to compare and improve many analytical and statistical methods relevant to microarray data analysis, including image processing, normalization, error modeling, combining of multiple reporters per gene, use of replicate experiments, and sample referencing schemes in measurements based on expression change. AVAILABILITY/SUPPLEMENTARY INFORMATION: Expression data and supplementary information are available at http://www.rii.com/publications/2003/HE_SDS.htm