Jaroslav Budiš

Utilization of benchtop next generation sequencing platforms ion torrent PGM and miseq in noninvasive prenatal testing for chromosome 21 trisomy and testing of impact of in silico and physical size selection on its analytical performance

Objectives The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. Methods Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. Results Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly—p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. Conclusions Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.

Frontal Cortical Atrophy as a Predictor of Poststroke Apathy

The aim of the study was to identify associations between the symptoms of poststroke apathy and sociodemographic, stroke-related (severity of stroke, degree of disability, and performance in activities of daily living), and radiological correlates. We determined the degree of cortical and subcortical brain atrophy, the severity of white matter and basal ganglia lesions on baseline computed tomography (CT) scans, and the localization of acute ischemia on control CT or magnetic resonance imaging scans in subacute stages of stroke. During follow-up examinations, in addition to the assessment of apathy symptoms using the Apathy Scale, we also evaluated symptoms of depression and anxiety using the Hospital Anxiety and Depression Scale. The study included 47 consecutive patients with acute ischemic stroke. Correlates significantly associated with apathy, determined at baseline and during follow-up, were entered into the “predictive” and “associative” multiple regression models, respectively. Frontal cortical atrophy and symptoms of depression were most strongly associated with poststroke apathy symptoms. In order to model an interrelation between both cortical atrophy and white matter lesions and aging, we supplemented 2 additional “predictive” models using interaction variables, whereby we confirmed the role of frontal cortical atrophy as a predictor of poststroke apathy also as a function of the increasing age of patients.

Innovative method for reducing uninformative calls in non-invasive prenatal testing

Motivation Non-invasive prenatal testing or NIPT is currently among the top researched topic in obstetric care. While the performance of the current state-of-the-art NIPT solutions achieve high sensitivity and specificity, they still struggle with a considerable number of samples that cannot be concluded with certainty. Such uninformative results are often subject to repeated blood sampling and re-analysis, usually after two weeks, and this period may cause a stress to the future mothers as well as increase the overall cost of the test. Results We propose a supplementary method to traditional z-scores to reduce the number of such uninformative calls. The method is based on a novel analysis of the length profile of circulating cell free DNA which compares the change in such profiles when random-based and length-based elimination of some fragments is performed. The proposed method is not as accurate as the standard z-score; however, our results suggest that combination of these two independent methods correctly resolves a substantial portion of healthy samples with an uninformative result. Additionally, we discuss how the proposed method can be used to identify maternal aberrations, thus reducing the risk of false positive and false negative calls. Availability and Implementation A particular implementation of the proposed methods is not provided with the manuscript. Contact Correspondence regarding the manuscript should be directed at Frantisek Duris (fduris@dcs.fmph.uniba.sk). Supplementary Information No additional supplementary information is available.

Non-invasive prenatal testing as a valuable source of population specific allelic frequencies

Low-coverage massively parallel genome sequencing for non-invasive prenatal testing (NIPT) of common aneuploidies is one of the most rapidly adopted and relatively low-cost DNA tests. Since aggregation of reads from a large number of samples allows overcoming the problems of extremely low coverage of individual samples, we describe the possible re-use of the data generated during NIPT testing for genome scale population specific frequency determination of small DNA variants, requiring no additional costs except of those for the NIPT test itself. We applied our method to a data set comprising of 1,548 original NIPT test results and evaluated the findings on different levels, from in silico population frequency comparisons up to wet lab validation analyses using a gold-standard method. The revealed high reliability of variant calling and allelic frequency determinations suggest that these NIPT data could serve as valuable alternatives to large scale population studies even for smaller countries around the world.

Dante: genotyping of known complex and expanded short tandem repeats

Motivation Short tandem repeats (STRs) are stretches of repetitive DNA in which short sequences, typically made of 2‐6 nucleotides, are repeated several times. Since STRs have many important biological roles and also belong to the most polymorphic parts of the human genome, they became utilized in several molecular‐genetic applications. Precise genotyping of STR alleles, therefore, was of high relevance during the last decades. Despite this, massively parallel sequencing (MPS) still lacks the analysis methods to fully utilize the information value of STRs in genome scale assays. Results We propose an alignment‐free algorithm, called Dante, for genotyping and characterization of STR alleles at user‐specified known loci based on sequence reads originating from STR loci of interest. The method accounts for natural deviations from the expected sequence, such as variation in the repeat count, sequencing errors, ambiguous bases and complex loci containing several different motifs. In addition, we implemented a correction for copy number defects caused by the polymerase induced stutter effect as well as a prediction of STR expansions that, according to the conventional view, cannot be fully captured by inherently short MPS reads. We tested Dante on simulated datasets and on datasets obtained by targeted sequencing of protein coding parts of thousands of selected clinically relevant genes. In both these datasets, Dante outperformed HipSTR and GATK genotyping tools. Furthermore, Dante was able to predict allele expansions in all tested clinical cases. Availability and implementation Dante is open source software, freely available for download at https://github.com/jbudis/dante. Supplementary information Supplementary data are available at Bioinformatics online.

Combining count- and length-based z-scores leads to improved predictions in non-invasive prenatal testing

Motivation Non‐invasive prenatal testing or NIPT is currently among the top researched topic in obstetric care. While the performance of the current state‐of‐the‐art NIPT solutions achieve high sensitivity and specificity, they still struggle with a considerable number of samples that cannot be concluded with certainty. Such uninformative results are often subject to repeated blood sampling and re‐analysis, usually after two weeks, and this period may cause a stress to the future mothers as well as increase the overall cost of the test. Results We propose a supplementary method to traditional z‐scores to reduce the number of such uninformative calls. The method is based on a novel analysis of the length profile of circulating cell free DNA which compares the change in such profiles when random‐based and length‐based elimination of some fragments is performed. The proposed method is not as accurate as the standard z‐score; however, our results suggest that combination of these two independent methods correctly resolves a substantial portion of healthy samples with an uninformative result. Additionally, we discuss how the proposed method can be used to identify maternal aberrations, thus reducing the risk of false positive and false negative calls. Availability and implementation The open‐source code of the proposed methods, together with test data, is freely available for non‐commercial users at github web page https://github.com/jbudis/lambda. Supplementary information Supplementary materials are available at Bioinformatics online.

Metagenomics of a nickel-resistant bacterial community in an anthropogenic nickel-contaminated soil in southwest Slovakia

Abstract The sampling sites situated in southwest Slovakia are according to environmental monitoring of Slovakia a part of strongly disturbed environment by heavy metals, mainly by high nickel concentrations. The aim of the present study was to characterise a complete microbial assemblage from a dump containing heavy-metal-contaminated waste as well as from farmland situated nearby this dump by using shotgun sequencing of 16S rDNA amplicons. It was found that nickel influenced both species richness and diversity and that microbiota of both samples differed significantly (Bray-Curtis dissimilarity 0.73) at genus level mainly by abundances of sequences from particular genera and occurrences of the unique genera in individual bacterial communities. In spite of these differences between microbial assemblages, both samples shared many bacterial genera that might constitute the specific nickel-resistant bacterial niche, and it was possible to delineate the core microbiome of our two samples at species level. The core set of 30 species, represented by the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria, suggest that these species might form a “core microbiome” of the specific nickel-resistant bacterial niche.