Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins.

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Diagnostic accuracy of random massively parallel sequencing for non-invasive prenatal detection of common autosomal aneuploidies: a collaborative study in Europe.

OBJECTIVE: The objective of this study is to validate the diagnostic accuracy of a non-invasive prenatal test for detecting trisomies 13, 18, and 21 for a population in Germany and Switzerland. METHODS: Random massively parallel sequencing was applied using Illumina sequencing platform HiSeq2000. Fetal aneuploidies were identified using a median absolute deviation based z-score equation. A bioinformatics algorithm based on guanine-cytosine normalization was applied after the data were unblinded. Results of massively parallel sequencing and invasive procedures were compared. RESULTS: Overall, 40/42 samples were correctly classified as trisomy 21-positive, including a translocation trisomy 21 [46,XY,der(13;21),+21] and a structural aberration of chromosome 21 [46,XX,rec(21)dup(21q)inv(21)(p12q21.1)] but not including a low percentage mosaic trisomy 21 [47,XY,+21/46,XY], [sensitivity: 95.2%; one-sided lower confidence limit: 85.8%]; 430/430 samples were correctly classified as trisomy 21-negative (specificity: 100%; one-sided lower CL: 99.3%). Using a new bioinformatics algorithm with guanine-cytosine normalization, detection of trisomy 21 was facilitated, and five of five trisomy 13 cases and eight of eight trisomy 18 cases were correctly identified. CONCLUSION: Our newly established non-invasive prenatal test allows detection of fetal trisomies 13, 18, and 21 with high accuracy in a population in Germany and Switzerland.

Species and condition specific adaptation of the transcriptional landscapes in Candida albicans and Candida dubliniensis.

BACKGROUND: Although Candida albicans and Candida dubliniensis are most closely related, both species behave significantly different with respect to morphogenesis and virulence. In order to gain further insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. RESULTS: Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. CONCLUSIONS: Species-specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level.

A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis.

Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human pathogen Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP-Seq and RNA-Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.

An Interspecies Regulatory Network Inferred from Simultaneous RNA-seq of Candida albicans Invading Innate Immune Cells.

The ability to adapt to diverse micro-environmental challenges encountered within a host is of pivotal importance to the opportunistic fungal pathogen Candida albicans. We have quantified C. albicans and M. musculus gene expression dynamics during phagocytosis by dendritic cells in a genome-wide, time-resolved analysis using simultaneous RNA-seq. A robust network inference map was generated from this dataset using NetGenerator, predicting novel interactions between the host and the pathogen. We experimentally verified predicted interdependent sub-networks comprising Hap3 in C. albicans, and Ptx3 and Mta2 in M. musculus. Remarkably, binding of recombinant Ptx3 to the C. albicans cell wall was found to regulate the expression of fungal Hap3 target genes as predicted by the network inference model. Pre-incubation of C. albicans with recombinant Ptx3 significantly altered the expression of Mta2 target cytokines such as IL-2 and IL-4 in a Hap3-dependent manner, further suggesting a role for Mta2 in host-pathogen interplay as predicted in the network inference model. We propose an integrated model for the functionality of these sub-networks during fungal invasion of immune cells, according to which binding of Ptx3 to the C. albicans cell wall induces remodeling via fungal Hap3 target genes, thereby altering the immune response to the pathogen. We show the applicability of network inference to predict interactions between host-pathogen pairs, demonstrating the usefulness of this systems biology approach to decipher mechanisms of microbial pathogenesis.

After the bottleneck: Genome-wide diversification of the Mycobacterium tuberculosis complex by mutation, recombination, and natural selection.

Many of the most virulent bacterial pathogens show low genetic diversity and sexual isolation. Accordingly, Mycobacterium tuberculosis, the deadliest human pathogen, is thought to be clonal and evolve by genetic drift. Yet, its genome shows few of the concomitant signs of genome degradation. We analyzed 24 genomes and found an excess of genetic diversity in regions encoding key adaptive functions including the type VII secretion system and the ancient horizontally transferred virulence-related regions. Four different approaches showed evident signs of recombination in M. tuberculosis. Recombination tracts add a high density of polymorphisms, and many are thus predicted to arise from outside the clade. Some of these tracts match Mycobacterium canettii sequences. Recombination introduced an excess of non-synonymous diversity in general and even more in genes expected to be under positive or diversifying selection, e.g., cell wall component genes. Mutations leading to non-synonymous SNPs are effectively purged in MTBC, which shows dominance of purifying selection. MTBC mutation bias toward AT nucleotides is not compensated by biased gene conversion, suggesting the action of natural selection also on synonymous changes. Together, all of these observations point to a strong imprint of recombination and selection in the genome affecting both non-synonymous and synonymous positions. Hence, contrary to some other pathogens and previous proposals concerning M. tuberculosis, this lineage may have come out of its ancestral bottleneck as a very successful pathogen that is rapidly diversifying by the action of mutation, recombination, and natural selection.

Pseudomonas putida KT2440 genome update by cDNA sequencing and microarray transcriptomics.

Pseudomonas putida KT2440 is a completely sequenced biosafety strain that has retained its capability to survive and function in the environment. The global mRNA expression profiles of the KT2440 strain grown at 10°C and 30°C were determined by deep cDNA sequencing to refine the genome annotation. Transcriptome sequencing identified 36 yet unknown small non-coding RNAs, 143 novel ORFs in 106 intergenic regions, 42 unclassified genes and eight highly expressed leaderless mRNA transcripts. The genome coordinates of eight genes and the organization of 57 operons were corrected. No overrepresented sequence motifs were detected in the 5'-untranslated regions. The 50 most highly expressed genes made up 60% of the total mRNA pool. Comparison of cDNA sequencing, Affymetrix and Progenika microarray data from the same mRNA preparation revealed a higher sensitivity and specificity of cDNA sequencing, a relatively poor correlation between the normalized cDNA reads and microarray signal intensities, and a systematic signal-dependent bias of microarrays in the detection of differentially regulated genes. The study demonstrates the power of next-generation cDNA sequencing for the quantitation of mRNA transcripts and the refinement of bacterial genome annotation.

Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains.

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.