Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening.

OBJECTIVES: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. METHODS: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. RESULTS: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. CONCLUSIONS: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Effects of drought and pluvial periods on fish and zooplankton communities in prairie lakes: systematic and asystematic responses.

The anticipated impacts of climate change on aquatic biota are difficult to evaluate because of potentially contrasting effects of temperature and hydrology on lake ecosystems, particularly those closed-basin lakes within semiarid regions. To address this shortfall, we quantified decade-scale changes in chemical and biological properties of 20 endorheic lakes in central North America in response to a pronounced transition from a drought to a pluvial period during the early 21st century. Lakes exhibited marked temporal changes in chemical characteristics and formed two discrete clusters corresponding to periods of substantially different effective moisture (as Palmer Drought Severity Index, PDSI). Discriminant function analysis (DFA) explained 90% of variability in fish assemblage composition and showed that fish communities were predicted best by environmental conditions during the arid interval (PDSI <-2). DFA also predicted that lakes could support more fish species during pluvial periods, but their occurrences may be limited by periodic stress due to recurrent droughts and physical barriers to colonization. Zooplankton taxonomic assemblages in fishless lakes were resilient to short-term changes in meteorological conditions, and did not vary between drought and deluge periods. Conversely, zooplankton taxa in fish-populated lakes decreased substantially in biomass during the wet interval, likely due to increased zooplanktivory by fish. The powerful effects of such climatic variability on hydrology and the strong subsequent links to water chemistry and biota indicate that future changes in global climate could result in significant restructuring of aquatic communities. Together these findings suggest that semiarid lakes undergoing temporary climate shifts provide a useful model system for anticipating the effects of global climate change on lake food webs.

A Systematic Method for Detecting Abnormal mRNA Splicing and Assessing Its Clinical Impact in Individuals Undergoing Genetic Testing for Hereditary Cancer Syndromes.

Nearly 14% of disease-causing germline variants result from the disruption of mRNA splicing. Most (67%) DNA variants predicted in silico to disrupt splicing are classified as variants of uncertain significance. An analytic workflow-splice effect event resolver (SPEER)-was developed and validated to use mRNA sequencing to reveal significant deviations in splicing, pinpoint the DNA variants potentially involved, and measure the deleterious effects of the altered splicing on mRNA transcripts, providing evidence for assessing the pathogenicity of the variant. SPEER was used to analyze leukocyte RNA encoding 63 hereditary cancer syndrome-related genes in 20,317 patients. Among 3563 patients (17.5%) with at least one DNA variant predicted to affect splicing, 971 (4.8%) had altered splicing with a deleterious effect on the transcript, and 40 had altered splicing due to a DNA variant located outside of the reportable range of the test. Integrating SPEER results into the interpretation of variants allowed variants of uncertain significance to be reclassified as pathogenic or likely pathogenic in 0.4%, and as benign or likely benign in 5.9%, of the 20,317 patients. SPEER-based evidence was associated with a significantly greater effect on classifications of pathogenic or likely pathogenic and benign or likely benign in nonwhite versus non-Hispanic white patients, illustrating that evidence derived from mRNA splicing analysis may help to reduce ethnic/ancestral disparities in genetic testing.

Assessing Limit of Detection in Clinical Sequencing.

Clinical reporting of solid tumor sequencing requires reliable assessment of the accuracy and reproducibility of each assay. Somatic mutation variant allele fractions may be below 10% in many samples due to sample heterogeneity, tumor clonality, and/or sample degradation in fixatives such as formalin. The toolkits available to the clinical sequencing community for correlating assay design parameters with assay sensitivity remain limited, and large-scale empirical assessments are often relied upon due to the lack of clear theoretical grounding. To address this uncertainty, a theoretical model was developed for predicting the expected variant calling sensitivity for a given library complexity and sequencing depth. Binomial models were found to be appropriate when assay sensitivity was only limited by library complexity or sequencing depth, but functional scaling for library complexity was necessary when both library complexity and sequencing depth were co-limiting. This model was empirically validated with sequencing experiments by using a series of DNA input amounts and sequencing depths. Based on these findings, a workflow is proposed for determining the limiting factors to sensitivity in different assay designs, and the formulas for these scenarios are presented. The approach described here provides designers of clinical assays with the methods to theoretically predict assay design outcomes a priori, potentially reducing burden in clinical tumor assay design and validation efforts.

Use of Treatment-Focused Tumor Sequencing to Screen for Germline Cancer Predisposition.

Next-generation sequencing assays are capable of identifying cancer patients eligible for targeted therapies and can also detect germline variants associated with increased cancer susceptibility. However, these capabilities have yet to be routinely harmonized in a single assay because of challenges with accurately identifying germline variants from tumor-only data. We have developed the Oncology and Hereditary Cancer Program targeted capture panel, which uses tumor tissue to simultaneously screen for both clinically actionable solid tumor variants and germline variants across 45 genes. Validation using 14 tumor specimens, composed of patient samples and cell lines analyzed in triplicate, demonstrated high coverage with sensitive and specific identification of single-nucleotide variants and small insertions and deletions. Average coverage across all targets remained >2000× in 198 additional patient tumor samples. Analysis of 55 formalin-fixed, paraffin-embedded tumor samples for the detection of known germline variants within a subset of cancer-predisposition genes, including one multiexon deletion, yielded a 100% detection rate, demonstrating that germline variants can be reliably detected in tumor samples using a single panel. Combining targetable somatic and actionable germline variants into a single tumor tissue assay represents a streamlined approach that can inform treatment for patients with advanced cancers as well as identify those with potential germline variants who are eligible for confirmatory testing, but would not otherwise have been identified.

Sample Tracking Using Unique Sequence Controls.

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.

Fixation Effects on Variant Calling in a Clinical Resequencing Panel.

Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. Several parameters relating to sequencing quality and variant calling were examined and quantified in tumor and normal colon epithelial tissues. We identified an alternative fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathologic review.

Ectomycorrhizal hyphae structure components of the soil bacterial community for decreased phosphatase production.

Ectomycorrhizal fungi (EMF) provide nutrients to their hosts by means of hyphae that extend beyond nutrient-depleted rhizosphere soil. Soil bacteria may compete with EMF for nutrients or may act synergistically to enhance nutrient supply to hosts. To assess the interactions between hyphae and bacteria, two types of small, sand-filled mesh bags were incubated in a Pseudotsuga menziesii/Betula papyrifera forest. The bags allowed ingrowth by EMF (35-µm mesh) or excluded hyphae (0.5-µm mesh), while allowing migration of soil bacteria. After incubation, bacteria were isolated from bags using a method to enrich for Gram-positive bacteria. Isolates were assayed for phosphatase and N-acetyl glucosaminidase (NAGase) activities to assess the potential to access organic phosphorus and nitrogen. The average phosphatase activities were higher in exclusion than ingrowth bags, while NAGase activities did not differ. Streptomyces isolates, which are expected to be strong competitors and antagonists of EMF, were more prevalent in ingrowth bags and yet had lower phosphatase activities. Furthermore, there were no indications of antagonism between fungi and Streptomyces, as there were no increases in NAGase activities in ingrowth bags. We conclude that fungal hyphae can structure components of the soil bacterial community for decreased extracellular enzyme production.