Wenying Pan

cisRED: a database system for genome-scale computational discovery of regulatory elements.

We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (). The database and high-throughput predictive pipeline are designed to address diverse target genomes in the context of rapidly evolving data resources and tools. Motifs are predicted in promoter regions using multiple discovery methods applied to sequence sets that include corresponding sequence regions from vertebrates. We estimate motif significance by applying discovery and post-processing methods to randomized sequence sets that are adaptively derived from target sequence sets, retain motifs with p-values below a threshold and identify groups of similar motifs and co-occurring motif patterns. The database offers information on atomic motifs, motif groups and patterns. It is web-accessible, and can be queried directly, downloaded or installed locally.

Noninvasive in vivo monitoring of tissue-specific global gene expression in humans.

Significance Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape of circulating RNA in human subjects. By focusing on tissue-specific genes, we were able to identify the relative contributions of these tissues to circulating RNA and monitor changes during tissue development and neurodegenerative disease states. Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer’s disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

High-performance binary protein interaction screening in a microfluidic format.

The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. For this, synergistic effects between the micro- and chemical architecture of the chip are exploited. The presented strategy of reducing the runtime rather than size and volume of the mechanical elements and biological reagent compartments will, in general, be of importance for future analytical test systems on microfluidic chips to overcome performance barriers.

Humans are colonized by many uncharacterized and highly divergent microbes

Blood circulates throughout the entire body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analysing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing

BACKGROUND Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods. METHODS We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women. RESULTS cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy. CONCLUSIONS Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.

Noninvasive blood tests for fetal development predict gestational age and preterm delivery

Toward more predictable birthdays Low-cost methods for monitoring fetal development could improve prenatal care, especially in low-resource settings. By measuring the levels of certain placental RNA transcripts in maternal blood, Ngo et al. developed two noninvasive blood tests that provide a window into the progression of individual pregnancies. In a small proof-of-concept study, the first blood test predicted fetal age and delivery date with an accuracy comparable to that of ultrasound. The second blood test, also examined in a small pilot study, discriminated women at risk of preterm delivery from those who delivered at full term. The next step will be to assess the reliability of the tests in large, blinded clinical trials. Science, this issue p. 1133 In pilot studies of pregnant women, RNA-based tests of maternal blood predicted delivery date and risk of early childbirth. Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (25 full-term and 13 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.

Recurrently Mutated Genes Differ between Leptomeningeal and Solid Lung Cancer Brain Metastases

&NA; When compared with solid brain metastases from NSCLC, leptomeningeal disease (LMD) has unique growth patterns and is rapidly fatal. Patients with LMD do not undergo surgical resection, limiting the tissue available for scientific research. In this study we performed whole exome sequencing on eight samples of LMD to identify somatic mutations and compared the results with those for 26 solid brain metastases. We found that taste 2 receptor member 31 gene (TAS2R31) and phosphodiesterase 4D interacting protein gene (PDE4DIP) were recurrently mutated among LMD samples, suggesting involvement in LMD progression. Together with a retrospective review of the charts of an additional 44 patients with NSCLC LMD, we discovered a surprisingly low number of KRAS mutations (n = 4 [7.7%]) but a high number of EGFR mutations (n = 33 [63.5%]). The median interval for development of LMD from NSCLC was shorter in patients with mutant EGFR (16.3 months) than in patients with wild‐type EGFR (23.9 months) (p = 0.017). Targeted analysis of recurrent mutations thus presents a useful complement to the existing diagnostic tool kit, and correlations of EGFR in LMD and KRAS in solid metastases suggest that molecular distinctions or systemic treatment pressure underpin the differences in growth patterns within the brain.

A Noninvasive Molecular Clock for Fetal Development Predicts Gestational Age and Preterm Delivery

We performed a high time-resolution, longitudinal study of normal pregnancy development by measuring cell-free RNA (cfRNA) in blood from women during each week of pregnancy. Analysis of tissue-specific transcripts in these samples enabled us to follow fetal and placental development with high resolution and sensitivity, and also to detect gene-specific responses of the maternal immune system to pregnancy. We established a “clock” for normal pregnancy development and enabled a direct molecular approach to determine expected delivery dates with comparable accuracy to ultrasound, creating the basis for a portable, inexpensive fetal dating method. We also identified a related gene set that accurately discriminated women at risk for spontaneous preterm delivery up to two months in advance of labor, forming the basis of a potential screening test for risk of preterm delivery.

Genomic Approaches to the Analysis of Cell Free Nucleic Acids

Recent advances in high throughput DNA sequencing and microarray technologies have revolutionized the field of genomics and also opened up many opportunities for the analysis of cell-free nucleic acids. These genomic approaches have not only provided a more comprehensive portrait of the landscape of cell-free nucleic acids, but also enabled a number of non-invasive genome-wide diagnostic methods. In this chapter, we introduce the basic mechanism of high throughput DNA sequencing and discuss some unique characteristics of cell-free nucleic acids that make their experimental procedure for high throughput analysis different from ordinary cellular nucleic acids. We describe different DNA sequencing protocols that have been used for cell-free DNA, including whole genome sequencing, exome sequencing and targeted amplicon sequencing. We explain the statistical model underlying the detection of copy number variation and point mutation from cell-free DNA. We also review recent clinical applications of sequencing cell-free DNA, from the non-invasive diagnosis of fetal genetic defects, to detection of tumor mutations from plasma and monitoring rejection of organ transplantation. In addition, we outline the perspective of profiling cell-free mRNA and cell-free microRNA using RNA-seq and microarray, and their potential applications. Finally, we conclude with discussions of the current challenges and possible future advances for genomic analysis of cell-free nucleic acids.