Dayne L. Filer

Objective Sequencing cell-free DNA now allows detection of large chromosomal abnormalities and dominant Mendelian disorders in the prenatal period. Improving upon these methods would allow newborn screening programs to begin with prenatal genetics, ultimately improving the management of rare genetic disorders. Methods As a pilot study, we performed exome sequencing on the cell-free DNA from three mothers with singleton pregnancies to assess the viability of broad sequencing modalities in a noninvasive prenatal setting. Results We found poor resolution of maternal and fetal genotypes due to both sampling and technical issues. Conclusion We find broad sequencing modalities inefficient for noninvasive prenatal applications. Alternatively, we suggest a more targeted path forward for noninvasive prenatal genotyping.

Background: As exome sequencing (ES) integrates into clinical practice, we should make every effort to utilize all information generated. Copy-number variation can lead to Mendelian disorders, but small copy-number variants (CNVs) often get overlooked or obscured by under-powered data collection. Many groups have developed methodology for detecting CNVs from ES, but existing methods often perform poorly for small CNVs and rely on large numbers of samples not always available to clinical laboratories. Furthermore, methods often rely on Bayesian approaches requiring user-defined priors in the setting of insufficient prior knowledge. This report first demonstrates the benefit of multiplexed exome capture (pooling samples prior to capture), then presents a novel detection algorithm, mcCNV (“multiplexed capture CNV”), built around multiplexed capture. Results: We demonstrate: (1) multiplexed capture reduces inter-sample variance; (2) our mcCNV method, a novel depth-based algorithm for detecting CNVs from multiplexed capture ES data, improves the detection of small CNVs. We contrast our novel approach, agnostic to prior information, with the the commonly-used ExomeDepth. In a simulation study mcCNV demonstrated a favorable false discovery rate (FDR). When compared to calls made from matched genome sequencing, we find the mcCNV algorithm performs comparably to ExomeDepth. Conclusion: Implementing multiplexed capture increases power to detect single-exon CNVs. The novel mcCNV algorithm may provide a more favorable FDR than ExomeDepth. The greatest benefits of our approach derive from (1) not requiring a database of reference samples and (2) not requiring prior information about the prevalance or size of variants.

What’s already known about this topic?: Sequencing-based noninvasive testing can detect large copy number abnormalities and some auto-somal dominant single-gene disorders Exome sequencing (ES) on fetal samples provides 20% diagnostic yield for structural abnormalities after normal karyotype & microarray What does this study add?: ES on cell-free DNA in three gravid patients with suspected genetic disease in the fetus We demonstrate broad sequencing approaches are limited by sampling and technical difficulties, concluding broad sequencing is currently inappropriate for noninvasive testing

Regularized regression analysis is a mature analytic approach to identify weighted sums of variables that predict outcomes. Typically, the number of subjects (N) is smaller than the number of predictors (p). Here, we present a novel coarse approximation linear function (CALF) to frugally select important predictors, build linear models, and discover causality. CALF is a linear regression strategy applied to normalized data that employs only a few (such as 2 to 20) nonzero weights, each +1 or -1. Metrics can be Welch t-test p-value, area under curve (AUC) of receiver operating characteristic, or Pearson correlation, depending upon data type and user preferences. For quantitative approximations, a linear fit (adding an intercept value and rescaling ±1 weights by a common multiplier) can be added to optimize mean squared error (MSE). Real medical data of five types were used to generate examples with goals of binary classification or real variable approximation. Predictors considered were real, sets of integers, or ternary values of single nucleotide polymorphisms. When applied to real data, CALF approximations outperformed in p-value, AUC, correlation, or MSE a popular regularized linear regression algorithm, namely, basic LASSO. It appears that using LASSO without considering CALF might risk wasting resources. Availability: R version: Comprehensive R Archive (CRAN): https://cran.r-project.org/web/packages/CALF/index.html Python 3.x version: GitHub: https://github.com/jorufo/CALF_Python Contact: clark_jeffries@med.unc.edu

Background: Tanzania’s Zanzibar archipelago has made significant gains in malaria control over the last decade and is a target for malaria elimination. Despite consistent implementation of effective tools since 2002, elimination has not been achieved. Importation of parasites from outside of the archipelago is thought to be an important cause of malaria’s persistence, but this paradigm has not been studied using modern genetic tools. Methods: We used whole-genome sequencing (WGS) to investigate the impact of importation, employing population genetic analyses of Plasmodium falciparum isolates from both the archipelago and mainland Tanzania. We assessed ancestry, levels of genetic diversity and differentiation, patterns of relatedness, and patterns of selection between these two populations by leveraging recent advances in deconvolution of genomes from polyclonal malaria infections. Results: We identified significant decreases in the effective population sizes in both populations in the timeframe of decreasing malaria transmission in Tanzania. Identity by descent analysis showed that parasites in the two populations shared large sections of their genomes, on the order of 5 cM, suggesting shared ancestry within the last 10 generations. Even with limited sampling,, we demonstrate a pair of isolates between the mainland and Zanzibar that are related at the expected level of half-siblings, consistent with recent importation Conclusions: These findings suggest that importation plays an increasing role for malaria incidence on Zanzibar and demonstrate the value of genomic approaches for identifying corridors of parasite movement to the island.