An approach to comparing tiling array and high throughput sequencing technologies for genomic transcript mapping

<p>Abstract</p> <p>Background</p> <p>There are two main technologies for transcriptome profiling, namely, tiling microarrays and high-throughput sequencing. Recently there has been a tremendous amount of excitement about the latter because of the advent of next-generation sequencing technologies and its promises. Consequently, the question of the moment is how these two technologies compare. Here we attempt to develop an approach to do a fair comparison of transcripts identified from tiling microarray and MPSS sequencing data.</p> <p>Findings</p> <p>This comparison is a challenging task because the sequencing data is discrete while the tiling array data is continuous. We use the published rice and <it>Arabidopsis </it>datasets which provide currently best matched sets of arrays and sequencing experiments using a slightly earlier generation of sequencing, the MPSS tag sequencing technology. After scoring the arrays consistently in both the organisms, a first pass comparison reveals a surprisingly small overlap in transcripts of 22% and 66% respectively, in rice and <it>Arabidopsis</it>. However, when we do the analysis in detail, we find that this is an underestimate. In particular, when we map the probe intensities onto the sequencing tags and then look at their intensity distribution, we see that they are very similar to exons. Furthermore, restricting our comparison to only protein-coding gene loci revealed a very good overlap between the two technologies.</p> <p>Conclusion</p> <p>Our approach to compare genome tiling microarray and MPSS sequencing data suggests that there is actually a reasonable overlap in transcripts identified by the two technologies. This overlap is distorted by the scoring and thresholding in the tiling array scoring procedure.</p

An approach to compare genome tiling microarray and MPSS sequencing data for transcript mapping

We are correcting the abstract of our published article ([1]). The sentence that starts "We observe that 4.5% of MPSS tags...." was not scientifically complete in the original abstract, having only two of the four numbers required to describe a comparison of two technologies in two different organisms. The abstract below more accurately describes our findings, as documented in Figure 1 of the manuscript

Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin.

Expression of the cell-surface antigen CD10 has long been used to define the lymphoid commitment of human cells. Here we report a unique lymphoid-primed population in human bone marrow that was generated from hematopoietic stem cells (HSCs) before onset of the expression of CD10 and commitment to the B cell lineage. We identified this subset by high expression of the homing molecule L-selectin (CD62L). CD10(-)CD62L(hi) progenitors had full lymphoid and monocytic potential but lacked erythroid potential. Gene-expression profiling placed the CD10(-)CD62L(hi) population at an intermediate stage of differentiation between HSCs and lineage-negative (Lin(-)) CD34(+)CD10(+) progenitors. CD62L was expressed on immature thymocytes, and its ligands were expressed at the cortico-medullary junction of the thymus, which suggested a possible role for this molecule in homing to the thymus. Our studies identify the earliest stage of lymphoid priming in human bone marrow

Haemogenic endocardium contributes to transient definitive haematopoiesis.

Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors

Comparison and calibration of transcriptome data from RNA-Seq and tiling arrays

<p>Abstract</p> <p>Background</p> <p>Tiling arrays have been the tool of choice for probing an organism's transcriptome without prior assumptions about the transcribed regions, but RNA-Seq is becoming a viable alternative as the costs of sequencing continue to decrease. Understanding the relative merits of these technologies will help researchers select the appropriate technology for their needs.</p> <p>Results</p> <p>Here, we compare these two platforms using a matched sample of poly(A)-enriched RNA isolated from the second larval stage of <it>C. elegans</it>. We find that the raw signals from these two technologies are reasonably well correlated but that RNA-Seq outperforms tiling arrays in several respects, notably in exon boundary detection and dynamic range of expression. By exploring the accuracy of sequencing as a function of depth of coverage, we found that about 4 million reads are required to match the sensitivity of two tiling array replicates. The effects of cross-hybridization were analyzed using a "nearest neighbor" classifier applied to array probes; we describe a method for determining potential "black list" regions whose signals are unreliable. Finally, we propose a strategy for using RNA-Seq data as a gold standard set to calibrate tiling array data. All tiling array and RNA-Seq data sets have been submitted to the modENCODE Data Coordinating Center.</p> <p>Conclusions</p> <p>Tiling arrays effectively detect transcript expression levels at a low cost for many species while RNA-Seq provides greater accuracy in several regards. Researchers will need to carefully select the technology appropriate to the biological investigations they are undertaking. It will also be important to reconsider a comparison such as ours as sequencing technologies continue to evolve.</p

Transmembrane Protein Oxygen Content and Compartmentalization of Cells

Recently, there was a report that explored the oxygen content of transmembrane proteins over macroevolutionary time scales where the authors observed a correlation between the geological time of appearance of compartmentalized cells with atmospheric oxygen concentration. The authors predicted, characterized and correlated the differences in the structure and composition of transmembrane proteins from the three kingdoms of life with atmospheric oxygen concentrations in geological timescale. They hypothesized that transmembrane proteins in ancient taxa were selectively excluding oxygen and as this constraint relaxed over time with increase in the levels of atmospheric oxygen the size and number of communication-related transmembrane proteins increased. In summary, they concluded that compartmentalized and non-compartmentalized cells can be distinguished by how oxygen is partitioned at the proteome level. They derived this conclusion from an analysis of 19 taxa. We extended their analysis on a larger sample of taxa comprising 309 eubacterial, 34 archaeal, and 30 eukaryotic complete proteomes and observed that one can not absolutely separate the two groups of cells based on partition of oxygen in their membrane proteins. In addition, the origin of compartmentalized cells is likely to have been driven by an innovation than happened 2700 million years ago in the membrane composition of cells that led to the evolution of endocytosis and exocytosis rather than due to the rise in concentration of atmospheric oxygen

c-Met-Dependent Multipotent Labyrinth Trophoblast Progenitors Establish Placental Exchange Interface

SummaryThe placenta provides the interface for gas and nutrient exchange between the mother and the fetus. Despite its critical function in sustaining pregnancy, the stem/progenitor cell hierarchy and molecular mechanisms responsible for the development of the placental exchange interface are poorly understood. We identified an Epcamhi labyrinth trophoblast progenitor (LaTP) in mouse placenta that at a clonal level generates all labyrinth trophoblast subtypes, syncytiotrophoblasts I and II, and sinusoidal trophoblast giant cells. Moreover, we discovered that hepatocyte growth factor/c-Met signaling is required for sustaining proliferation of LaTP during midgestation. Loss of trophoblast c-Met also disrupted terminal differentiation and polarization of syncytiotrophoblasts, leading to intrauterine fetal growth restriction, fetal liver hypocellularity, and demise. Identification of this c-Met-dependent multipotent LaTP provides a landmark in the poorly defined placental stem/progenitor cell hierarchy and may help us understand pregnancy complications caused by a defective placental exchange

A critical appraisal of mucormycosis in COVID- 19 patients in a tertiary care centre in India

Background and Purpose: Morbidity and mortality of opportunistic fungal infections in COVID-19 patients are less studied and defined. The patients receiving immunosuppressive therapy, broad-spectrum antibiotics, corticosteroids, and invasive and non-invasive ventilation are the high-risk groups.Materials and Methods: The demographic profile as well as clinical and radiological findings of all the patients with COVID-19 suspected of Mucormycosis (MM) were recorded. The tissue samples from all the patients were sent for microbiological (KOH mount and culture) and histopathological analysis for confirmation of MM.Results: In total, 45 COVID-19 patients suspected of MM were included in the study an MM was confirmed in 42 patients. The mean age of the patients was 50.30±14.17 years with a female: male ratio of 1.1:1. The most common symptom was headache (52.38%) followed by purulent nasal discharge (38.09%) and facial pain in 33.33% of the cases. The ocular symptoms included a diminution of vision (33.33%) and redness of the eye (2.38%).The most common site of involvement was rhino-orbital (42.85%) followed by sinonasal (23.80%) and rhino cerebral (19.04%). Majority (38.09%) of the patients were diagnosed with stage II of Rhino-orbital-cerebral Mucormycosis (ROCM) based on radiology. A history of diabetes mellitus and steroids was present in 97.61% and 85.71% of the cases, respectively. Moreover, KOH was positive for MM in 97.61% of the cases while the culture was positive in only 35.71% of the cases. In addition, on histopathology, MM was confirmed in 64.28 % of the cases. Mixed growth with Aspergillus species and Rhizopus species was observed in 14.28% of the cases in culture and 11.90% of the cases in histopathology test. Furthermore, angioinvasion was found in 23.80% of the cases according to the histopathology test.Conclusion: Based on the results, the most common conditions associated with MM in COVID-19 patients were diabetes mellitus and steroid therapy. A high level of clinical suspicion aided with diagnostic tests, including KOH mount, culture, histopathology, and radiology which helped the early detection of opportunistic fungal infection in COVID-19 patients to ensure timely treatment

Scl binds to primed enhancers in mesoderm to regulate hematopoietic and cardiac fate divergence

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre-established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage-specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated