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.
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.
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. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nature methods frequencies of variants in our sample set and six different ExAC populations. Results are shown separately for (a) SNVs based on 68,326 variants; and (b) indels based on 2,909. In both cases, only those variants were used, which were simultaneously identified in each data subset with MAF higher than 5%. AFR = African/African American, AMR = American (Latino), EAS = East Asian, FIN = Finnish, NFE = Non-Finnish European, SAS = South Asian, SVK = Slovak. 2 7 SupplementaryTable 1 (uploaded as a separate Excel sheet of the Supplementary Table file):List of variants identified in the region of the CLCN1 (chloride voltage-gated channel 1, OMIM * 118425) gene in ExAC populations, dbSNP and in our data set. Only 17 of them had ExAC frequencies above 5% (highlighted in yellow). All but five were identified in our data set too with very similar calculated population frequencies. The exceptions, rs34904831, rs191902231, rs182668076, rs2280663 and rs73726622, were found to have ExAC frequencies +/-5% (depending on population). Originally three of these variants were identified in our data set too, although they were filtered out due slightly lower than 5% frequency (Suppl.Tab.1), further suggesting the feasibility of lowering our frequency restrictions. Our data contained also 89 variants missing from ExAC (highlighted in red), but found to have each of them deep intronic positions falling outside ExAC´s BED file. AFR = African/African American, AMR = American, EAS = East Asian, FIN = Finnish, NFE = Non-Finnish European, SAS = South Asian, SVK = Slovak. Supplementary Table 2 (uploaded as a separate Excel sheet of the Supplementary Table file): Verification of 87 positions in five polymorphic genomic loci of 58 randomly selected samples of our sample set from which genomic DNA was available to validation purposes. Reads from positions covered by multiple reads in individual samples (such in case of sample ID4730, where both alleles we...
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.
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