Cell-free DNA testing of an extended range of chromosomal anomalies: clinical experience with 6,388 consecutive cases

PURPOSE: Cell-free DNA (cfDNA) testing for fetal aneuploidies was broadly implemented for common trisomies and sex-chromosome anomalies (SCAs). However, such an approach identifies only 75 to 85% of clinically relevant aneuploidies. METHODS: We present a consecutive series of 6,388 cases, thus uncovering a broader array of aneuploidies, including the rare autosomal trisomies (RATs) and the maternally inherited deletion and duplication copy-number variations (CNVs), with complete and stratified follow-up by amniocentesis. Combined measurements of z-scores and the fetal fraction, in conjunction with fetal cfDNA enrichment, were used to stratify the likelihood of true and false results. RESULTS: We obtained an incremental diagnostic yield of 50%; RATs and CNVs were found to be significant causes of fetal pathology. Scrutinizing z-scores and the fetal fraction made it possible to distinguish the sources of false-negative results; predict the likelihood of false-positive results for major trisomies and SCAs; classify maternal mosaic SCAs and CNVs, preventing false-positive results; and robustly identify maternally inherited CNVs and detect recurrent genomic disorders as a standardized function of the fetal fraction. CONCLUSION: With the clinical pertinence of this broader detection scheme confirmed, we offer recommendations for its implementation. Genet Med 19 2, 169–175

Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei

Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/

Whole-genome shotgun sequence of pseudomonas viridiflava, a bacterium species pathogenic to ararabidopsis thaliana

We report here the first whole-genome shotgun sequence of Pseudomonas viridiflava strain UASWS38, a bacterium species pathogenic to the biological model plant Arabidopsis thaliana but also usable as a biological control agent and thus of great scientific interest for understanding the genetics of plant-microbe interactions

Whole-genome shotgun sequence of bacillus amyloliquefaciens strain UASWS BA1, a bacterium antagonistic to plant pathogenic fungi

We report here the whole-genome shotgun sequence of Bacillus amyloliquefaciens strain UASWS BA1, isolated from inner wood tissues of a decaying Platanus × acerifolia tree. This strain proved to be antagonistic to several plant pathogenic fungi and oomycetes and can be developed as a biological control agent in agriculture

Whole-genome shotgun sequence of arthrospira platensis strain paraca, a cultivated and edible cyanobacterium

Here we report the whole-genome shotgun sequence of a Peruvian strain of Arthrospira platensis (Paraca), a cultivated and edible haloalkaliphilic cyanobacterium of great scientific, technical, and economic potential

Metagenomic study of the oral microbiota by Illumina high-throughput sequencing

To date, metagenomic studies have relied on the utilization and analysis of reads obtained using 454 pyrosequencing to replace conventional Sanger sequencing. After extensively scanning the 16S ribosomal RNA (rRNA) gene, we identified the V5 hypervariable region as a short region providing reliable identification of bacterial sequences available in public databases such as the Human Oral Microbiome Database. We amplified samples from the oral cavity of three healthy individuals using primers covering an approximately 82-base segment of the V5 loop, and sequenced using the Illumina technology in a single orientation. We identified 135 genera or higher taxonomic ranks from the resulting 1,373,824 sequences. While the abundances of the most common phyla (Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria and TM7) are largely comparable to previous studies, Bacteroidetes were less present. Potential sources for this difference include classification bias in this region of the 16S rRNA gene, human sample variation, sample preparation and primer bias. Using an Illumina sequencing approach, we achieved a much greater depth of coverage than previous oral microbiota studies, allowing us to identify several taxa not yet discovered in these types of samples, and to assess that at least 30,000 additional reads would be required to identify only one additional phylotype. The evolution of high-throughput sequencing technologies, and their subsequent improvements in read length enable the utilization of different platforms for studying communities of complex flora. Access to large amounts of data is already leading to a better representation of sample diversity at a reasonable cost

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

<div><p>Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET^® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET^® <i>in vitro</i> performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.</p></div

<i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.

<p><i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.</p

Assessment of ISET^® intra-assay accuracy and precision.

<p>50 A549 cells were spiked into 5 mL of blood (n = 3 experiments, 43 to 49 cells per 5 mL). The number of tumor cells found on each spot after ISET^® filtration (each corresponding to the filtration of 1 mL of blood) was recorded. Experiments were done on 5 spots but for intra-assay precision and accuracy only the assessment of the number of cells found on single spots or randomly grouped spots (any 1, any 2, any 3, any 4 spots) is relevant. The cell counting on the combination of all the 5 spots was used as reference. Results show that cell counting on four spots exhibited a representative mean tumor cells value per mL of blood. (A) Bar chart with the mean tumor cell number per spot and corresponding standard error of the mean (error bars) depending on the number of spots analyzed. Error bars are calculated using the standard deviation in different combinations of any 4 spots, any 3 spots, any 2 spots or any 1 spot, respectively. If only one spot is considered, standard deviation is higher than when counting 4 spots, showing that counting on four spots gives a reliable mean tumor cells' value per mL of blood. (B) Table indicating the number of tumor cells found on each spot for each of the five experiments, the 95% confidence interval (CI), the precision (%CV) and the accuracy (%error) depending on the number of spots analyzed (1 to 4) as compared to the analysis on five spots.</p