Analysis of RNA virome in rectal swabs of healthy and diarrheic pigs of different age.

The porcine enteric virome comprises a wide range of eukaryotic and prokaryotic viruses in healthy and diarrheic pigs. As RNA viruses are considered to be important agents responsible for diarrhoea in pigs, this work was focused on the RNA virome. To identify viruses, a next generation sequencing technique and bioinformatics analysis was employed. A wide spectrum of viral genera with RNA genomes, such as Kobuvirus, Picobirnavirus, Teschovirus, Posavirus, Mamastovirus, Enterovirus and Rotavirus were identified in both diarrheic and healthy pigs. No clear differences in the virome composition were found between healthy and diarrheic pigs. The data visualisation using Non-Metric Multidimensional Scaling as well as by the Analysis of Similarities test suggested that the virome depended on the age of animals and differed in piglets when compared to weaned and fattening pigs.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions.

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.

Validation of Copy Number Variants Detection from Pregnant Plasma Using Low-Pass Whole-Genome Sequencing in Noninvasive Prenatal Testing-Like Settings.

Detection of copy number variants as an integral part of noninvasive prenatal testing is increasingly used in clinical practice worldwide. We performed validation on plasma samples from 34 pregnant women with known aberrations using cell-free DNA sequencing to evaluate the sensitivity for copy number variants (CNV) detection using an in-house CNV fraction-based detection algorithm. The sensitivity for CNVs smaller than 3 megabases (Mb), larger than 3Mb, and overall was 78.57%, 100%, and 90.6%, respectively. Regarding the fetal fraction, detection sensitivity in the group with a fetal fraction of less than 10% was 57.14%, whereas there was 100% sensitivity in the group with fetal fraction exceeding 10%. The assay is also capable of indicating whether the origin of an aberration is exclusively fetal or fetomaternal/maternal. This validation demonstrated that a CNV fraction-based algorithm was applicable and feasible in clinical settings as a supplement to testing for common trisomies 21, 18, and 13.

Result of Prospective Validation of the Trisomy Test® for the Detection of Chromosomal Trisomies.

Noninvasive prenatal testing (NIPT) is one of the most common prenatal screening tests used worldwide. Trisomy Test® belongs to NIPT tests based on low-coverage whole-genome sequencing. In our prospective study, 7279 samples of pregnant women collected during approximately two years were analyzed. In this cohort, 117 positive cases for trisomies 21, 18, and 13 were reported. An in-house designed bioinformatic pipeline and proprietary biostatistical approach was used for the detection of trisomies. The pooled sensitivity and specificity of our test reached 99.12% and 99.94%, respectively. The proportion of repeatedly uninformative results after repeated blood draws was 1.11%. Based on the presented results, we can confirm that the Trisomy Test® is fully comparable with other commercial NIPT tests available worldwide.

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 1501 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 based on Sanger sequencing. 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.

On the critical evaluation and confirmation of germline sequence variants identified using massively parallel sequencing.

Although massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes. Detailed examination of false-positive and false-negative findings revealed typical error sources connected to low read depth/coverage, incomplete reference genome, indel realignment problems, as well as microsatellite associated amplification errors leading to base miss-calling. However, all these error types were identifiable with thorough manual revision of aligned reads according to specific patterns of distributions of variants and their corresponding reads. Moreover, our results point to dependence of both basic quantitative metrics (such as total read counts, alternative allele read counts and allelic balance) together with specific genotype quality scores on the used bioinformatics pipeline, stressing thus the need for establishing of specific thresholds for these metrics in each laboratory and for each involved pipeline independently.

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.