Craig A. Struble

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

OBJECTIVE We 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. STUDY DESIGN We 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. RESULTS In 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. CONCLUSION Digital analysis of selected regions and FORTE enable accurate, scalable noninvasive fetal aneuploidy detection.

Overview of BioCreative II gene mention recognition

Nineteen teams presented results for the Gene Mention Task at the BioCreative II Workshop. In this task participants designed systems to identify substrings in sentences corresponding to gene name mentions. A variety of different methods were used and the results varied with a highest achieved F1 score of 0.8721. Here we present brief descriptions of all the methods used and a statistical analysis of the results. We also demonstrate that, by combining the results from all submissions, an F score of 0.9066 is feasible, and furthermore that the best result makes use of the lowest scoring submissions.

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.

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.

Transparent sharing of Java applets: a replicated approach

People interact together in all aspects of life and, as computers have become prevalent, users seek computer support for their interactions. The WWW provides an unprecedented opportunity for users to interact with each other, and the advent of JavaThfl has created a consistent computing environment to support synchronous collaboration. We describe JAMM, a prototype Java runtime environment that supports the shared use of existing Java applets, thus leveraging the existing base of software for synchronous collaboration. Our approach is based on a replicated architecture, where each user maintains their own copy of the Java applet, and the users’ input events are broadcast to each applet copy. We discuss solutions to certain key problems, such as unanticipated sharing, supporting late-joiners, and replicating input sources other than user inputs (e.g., files, sockets, and random number generators).

Bioaugmentation for Improved Recovery of Anaerobic Digesters After Toxicant Exposure

Bioaugmentation was investigated as a method to decrease the recovery period of anaerobic digesters exposed to a transient toxic event. Two sets of laboratory-scale digesters (SRT = 10 days, OLR = 2 g COD/L-day), started with inoculum from a digester stabilizing synthetic municipal wastewater solids (MW) and synthetic industrial wastewater (WW), respectively, were transiently exposed to the model toxicant, oxygen. Bioaugmented digesters received 1.2 g VSS/L-day of an H2-utilizing culture for which the archaeal community was analyzed. Soon after oxygen exposure, the bioaugmented digesters produced 25-60% more methane than non-bioaugmented controls (p < 0.05). One set of digesters produced lingering high propionate concentrations, and bioaugmentation resulted in significantly shorter recovery periods. The second set of digesters did not display lingering propionate, and bioaugmented digesters recovered at the same time as non-bioaugmented controls. The difference in the effect of bioaugmentation on recovery may be due to differences between microbial communities of the digester inocula originally employed. In conclusion, bioaugmentation with an H(2)-utilizing culture is a potential tool to decrease the recovery period, decrease propionate concentration, and increase biogas production of some anaerobic digesters after a toxic event. Digesters already containing rapidly adaptable microbial communities may not benefit from bioaugmentation, whereas other digesters with poorly adaptable microbial communities may benefit greatly.

Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters.

Accumulation of acids in anaerobic digesters after organic overload can inhibit or stop CH4 production. Therefore, methods to reduce acid concentrations would be helpful. One potential method to improve recovery involves bioaugmentation, addition of specific microorganisms to improve performance. In this study, transiently overloaded digesters were bioaugmented with a propionate-degrading enrichment culture in an effort to decrease recovery time. Biomass samples from 14 different, full-scale anaerobic digesters were screened for specific methanogenic activity (SMA) against propionate; the microbial communities were also compared. SMA values spanned two orders of magnitude. Principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns for a functional gene (mcrA) suggested an underlying community structure-activity relationship; the presence of hydrogenotrophic methanogens closely related to Methanospirillum hungatei and Methanobacterium beijingense was associated with high propionate SMA values. The biomass sample demonstrating the highest SMA was enriched for propionate degrading activity and then used to bioaugment overloaded digesters. Bioaugmented digesters recovered more rapidly following the organic overload, requiring approximately 25 days (2.5 solids retention times (SRTs)) less to recover compared to non-bioaugmented digesters. Benefits of bioaugmentation continued for more than 12 SRTs after organic overload. Bioaugmentation is a promising approach to decrease recovery time after organic overload.

Assessment of fetal sex chromosome aneuploidy using directed cell-free DNA analysis.

Objective: To examine the performance of chromosome-selective sequencing of cell-free (cf) DNA in maternal blood for assessment of fetal sex chromosome aneuploidies. Methods: This was a case-control study of 177 stored maternal plasma samples, obtained before fetal karyotyping at 11-13 weeks of gestation, from 59 singleton pregnancies with fetal sex chromosome aneuploidies (45,X, n = 49; 47,XXX, n = 6; 47,XXY, n = 1; 47,XYY, n = 3) and 118 with euploid fetuses (46,XY, n = 59; 46,XX, n = 59). Digital analysis of selected regions (DANSR™) on chromosomes 21, 18, 13, X and Y was performed and the fetal-fraction optimized risk of trisomy evaluation (FORTE™) algorithm was used to estimate the risk for non-disomic genotypes. Performance was calculated at a risk cut-off of 1:100. Results: Analysis of cfDNA provided risk scores for 172 (97.2%) samples; 4 samples (45,X, n = 2; 46,XY, n = 1; 46,XX, n = 1) had an insufficient fetal cfDNA fraction for reliable testing and 1 case (47,XXX) failed laboratory quality control metrics. The classification was correct in 43 (91.5%) of 47 cases of 45,X, all 5 of 47,XXX, 1 of 47,XXY and 3 of 47,XYY. There were no false-positive results for monosomy X. Discussion: Analysis of cfDNA by chromosome-selective sequencing can correctly classify fetal sex chromosome aneuploidy with reasonably high sensitivity.

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.