riboviz: analysis and visualization of ribosome profiling datasets.

BACKGROUND: Using high-throughput sequencing to monitor translation in vivo, ribosome profiling can provide critical insights into the dynamics and regulation of protein synthesis in a cell. Since its introduction in 2009, this technique has played a key role in driving biological discovery, and yet it requires a rigorous computational toolkit for widespread adoption. DESCRIPTION: We have developed a database and a browser-based visualization tool, riboviz, that enables exploration and analysis of riboseq datasets. In implementation, riboviz consists of a comprehensive and flexible computational pipeline that allows the user to analyze private, unpublished datasets, along with a web application for comparison with published yeast datasets. Source code and detailed documentation are freely available from https://github.com/shahpr/RiboViz . The web-application is live at www.riboviz.org. CONCLUSIONS: riboviz provides a comprehensive database and analysis and visualization tool to enable comparative analyses of ribosome-profiling datasets. This toolkit will enable both the community of systems biologists who study genome-wide ribosome profiling data and also research groups focused on individual genes to identify patterns of transcriptional and translational regulation across different organisms and conditions.

Evaluation of comprehensive chromosome screening platforms for the detection of mosaic segmental aneuploidy.

PURPOSE: A subset of preimplantation embryos identified as euploid may in fact possess both whole and sub-chromosomal mosaicism, raising concerns regarding the predictive value of current comprehensive chromosome screening (CCS) methods utilizing a single biopsy. Current CCS methods may be capable of detecting sub-chromosomal mosaicism in a trophectoderm biopsy by examining intermediate levels of segmental aneuploidy within a biopsy. This study evaluates the sensitivity and specificity of segmental aneuploidy detection by three commercially available CCS platforms utilizing a cell line mixture model of segmental mosaicism in a six-cell trophectoderm biopsy. METHODS: Two cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female cell line containing a 16.2 Mb deletion on chromosome 5 and a male cell line containing a 25.5 Mb deletion on chromosome 4 were used to create mixtures at each level. Six replicates of each mixture were prepared, randomized, and blinded for analysis by one of the three CCS platforms (SNP-array, VeriSeq NGS, or NexCCS). Sensitivity and specificity of segmental aneuploidy at each level of mosaicism was determined and compared between each platform. Additionally, an alternative VeriSeq NGS analysis method utilizing previously published criteria was evaluated. RESULTS: Examination of the default settings of each platform revealed that the sensitivity was significantly different between NexCCS and SNP up to 50% mosaicism, custom VeriSeq, and SNP-array up to 66% mosaicism, and between NexCCS and custom VeriSeq up to 50% mosaicism. However, no statistical difference was observed in mixtures with >50% mosaicism with any platform. No comparison was made between default VeriSeq, as it does not report segmental imbalances. Furthermore, while the use of previously published criteria for VeriSeq NGS significantly increased sensitivity at low levels of mosaicism, a significant decrease in specificity was observed (66% false positive prediction of segmental aneuploidy). CONCLUSION: These results demonstrate the potential of NGS-based detection methods to detect segmental mosaicism within a biopsy. However, these data also demonstrate that a balance between sensitivity and specificity should be more carefully considered. These results emphasize the importance of vigorous preclinical evaluation of new testing criteria prior to clinical implementation providing a point of departure for further algorithm development and improved detection of mosaicism within preimplantation embryos.

A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

PURPOSE: A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. METHODS: Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. RESULTS: With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). CONCLUSIONS: By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.

STUDY QUESTION: What is the prevalence and developmental significance of morphologic nuclear abnormalities in human preimplantation embryos? SUMMARY ANSWER: Nuclear abnormalities are commonly found in human IVF embryos and are associated with DNA damage, aneuploidy, and decreased developmental potential. WHAT IS KNOWN ALREADY: Early human embryonic development is complicated by genomic errors that occur after fertilization. The appearance of extra-nuclear DNA, which has been observed in IVF, may be a result of such errors. However, the mechanism by which abnormal nuclei form and the impact on DNA integrity and embryonic development is not understood. STUDY DESIGN, SIZE, DURATION: Cryopreserved human cleavage-stage embryos (n = 150) and cryopreserved blastocysts (n = 105) from clinical IVF cycles performed between 1997 and 2008 were donated for research. Fresh embryos (n = 60) of poor quality that were slated for discard were also used. Immunohistochemical, microscopic and cytogenetic analyses at different developmental stages and morphologic grades were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos were fixed and stained for DNA, centromeres, mitotic activity and DNA damage and imaged using confocal microscopy. Rates of abnormal nuclear formation were compared between morphologically normal cleavage-stage embryos, morphologically normal blastocysts, and poor quality embryos. To control for clinical and IVF history of oocytes donors, and quality of frozen embryos within our sample, cleavage-stage embryos (n = 52) were thawed and fixed at different stages of development and then analyzed microscopically. Cleavage-stage embryos (n = 9) were thawed and all blastomeres (n = 62) were disaggregated, imaged and analyzed for karyotype. Correlations were made between microscopic and cytogenetic findings of individual blastomeres and whole embryos. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of microscopic nuclear abnormalities was lower in blastocysts (5%; 177/3737 cells) than in cleavage-stage embryos (16%, 103/640 blastomeres, P < 0.05) and highest in arrested embryos (65%; 44/68 blastomeres, P < 0.05). DNA damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The embryos used were de-identified, and the clinical and IVF history was unknown. WIDER IMPLICATIONS OF THE FINDINGS: This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF. STUDY FUNDING/COMPETING INTERESTS: The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest.

Hydrogen sulfide improves drought tolerance in Arabidopsis thaliana by microRNA expressions.

Hydrogen sulfide (H2S) is a gasotransmitter and plays an important role in many physiological processes in mammals. Studies of its functions in plants are attracting ever growing interest, for example, its ability to enhance drought resistance in Arabidopsis. A general role of microRNAs (miRNAs) in plant adaptive responses to drought stress has thereby increased our interest to delve into the possible interplay between H2S and miRNAs. Our results showed that treating wild type (WT) Arabidopsis seedlings with polyethylene glycol 8000 (PEG8000) to simulate drought stress caused an increase in production rate of endogenous H2S; and a significant transcriptional reformation of relevant miRNAs, which were also triggered by exogenous H2S in WT. When lcd mutants (with lower H2S production rate than WT) were treated with PEG8000, they showed lower levels of miRNA expression changes than WT. In addition, we detected significant changes in target gene expression of those miRNAs and the corresponding phenotypes in lcd, including less roots, retardation of leaf growth and development and greater superoxide dismutase (SOD) activity under drought stress. We thereby conclude that H2S can improve drought resistance through regulating drought associated miRNAs in Arabidopsis.