High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.

PURPOSE: The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body mass index (and >5% overall) receive a test failure due to low FF (<4%). METHODS: A scalable fetal fraction amplification (FFA) technology was analytically validated on 1264 samples undergoing whole-genome sequencing (WGS)-based NIPS. All samples were tested with and without FFA. RESULTS: Zero samples had FF < 4% when screened with FFA, whereas 1 in 25 of these same patients had FF < 4% without FFA. The average increase in FF was 3.9-fold for samples with low FF (2.3-fold overall) and 99.8% had higher FF with FFA. For all abnormalities screened on NIPS, z-scores increased 2.2-fold on average in positive samples and remained unchanged in negative samples, powering an increase in NIPS sensitivity and specificity. CONCLUSION: FFA transforms low-FF samples into high-FF samples. By combining FFA with WGS-based NIPS, a single round of NIPS can provide nearly all women with confident results about the broad range of potential fetal chromosomal abnormalities across the genome.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

Detecting clinically actionable variants in the 3' exons of PMS2 via a reflex workflow based on equivalent hybrid capture of the gene and its pseudogene.

BACKGROUND: Hereditary cancer screening (HCS) for germline variants in the 3' exons of PMS2, a mismatch repair gene implicated in Lynch syndrome, is technically challenging due to homology with its pseudogene PMS2CL. Sequences of PMS2 and PMS2CL are so similar that next-generation sequencing (NGS) of short fragments-common practice in multigene HCS panels-may identify the presence of a variant but fail to disambiguate whether its origin is the gene or the pseudogene. Molecular approaches utilizing longer DNA fragments, such as long-range PCR (LR-PCR), can definitively localize variants in PMS2, yet applying such testing to all samples can have logistical and economic drawbacks. METHODS: To address these drawbacks, we propose and characterize a reflex workflow for variant discovery in the 3' exons of PMS2. We cataloged the natural variation in PMS2 and PMS2CL in 707 samples and designed hybrid-capture probes to enrich the gene and pseudogene with equal efficiency. For PMS2 exon 11, NGS reads were aligned, filtered using gene-specific variants, and subject to standard diploid variant calling. For PMS2 exons 12-15, the NGS reads were permissively aligned to PMS2, and variant calling was performed with the expectation of observing four alleles (i.e., tetraploid calling). In this reflex workflow, short-read NGS identifies potentially reportable variants that are then subject to disambiguation via LR-PCR-based testing. RESULTS: Applying short-read NGS screening to 299 HCS samples and cell lines demonstrated >99% analytical sensitivity and >99% analytical specificity for single-nucleotide variants (SNVs) and short insertions and deletions (indels), as well as >96% analytical sensitivity and >99% analytical specificity for copy-number variants. Importantly, 92% of samples had resolved genotypes from short-read NGS alone, with the remaining 8% requiring LR-PCR reflex. CONCLUSION: Our reflex workflow mitigates the challenges of screening in PMS2 and serves as a guide for clinical laboratories performing multigene HCS. To facilitate future exploration and testing of PMS2 variants, we share the raw and processed LR-PCR data from commercially available cell lines, as well as variant frequencies from a diverse patient cohort.

Group Testing Approach for Trinucleotide Repeat Expansion Disorder Screening.

BACKGROUND: Fragile X syndrome (FXS, OMIM #300624) is an X-linked condition caused by trinucleotide repeat expansions in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. FXS testing is commonly performed in expanded carrier screening and has been proposed for inclusion in newborn screening. However, because pathogenic alleles are long and have low complexity (>200 CGG repeats), FXS is currently tested by a single-plex electrophoresis-resolved PCR assay rather than multiplexed approaches like next-generation sequencing or mass spectrometry. In this work, we sought an experimental design based on nonadaptive group testing that could accurately and reliably identify the size of abnormally expanded FMR1 alleles of males and females. METHODS: We developed a new group testing scheme named StairCase (SC) that was designed to the constraints of the FXS testing problem, and compared its performance to existing group testing schemes by simulation. We experimentally evaluated SC's performance on 210 samples from the Coriell Institute biorepositories using pooled PCR followed by capillary electrophoresis on 3 replicates of each of 3 pooling layouts differing by the mapping of samples to pools. RESULTS: The SC pooled PCR approach demonstrated perfect classification of samples by clinical category (normal, intermediate, premutation, or full mutation) for 90 positives and 1800 negatives, with a batch of 210 samples requiring only 21 assays. CONCLUSIONS: Group testing based on SC is an implementable approach to trinucleotide repeat expansion disorder testing that offers ≥10-fold reduction in assay costs over current single-plex methods.

Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment.

The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy number variants (CNVs) in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). We also demonstrated the screen's high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

A BioBrick compatible strategy for genetic modification of plants.

BACKGROUND: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. RESULTS: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. CONCLUSIONS: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.