Ultraaccurate genome sequencing and haplotyping of single human cells.

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs

Uniaxial Mechanical Strain Modulates the Differentiation of Neural Crest Stem Cells into Smooth Muscle Lineage on Micropatterned Surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors

The photometric observation of the quasi-simultaneous mutual eclipse and occultation between Europa and Ganymede on 22 August 2021

Mutual events (MEs) are eclipses and occultations among planetary natural satellites. Most of the time, eclipses and occultations occur separately. However, the same satellite pair will exhibit an eclipse and an occultation quasi-simultaneously under particular orbital configurations. This kind of rare event is termed as a quasi-simultaneous mutual event (QSME). During the 2021 campaign of mutual events of jovian satellites, we observed a QSME between Europa and Ganymede. The present study aims to describe and study the event in detail. We observed the QSME with a CCD camera attached to a 300-mm telescope at the Hong Kong Space Museum Sai Kung iObservatory. We obtained the combined flux of Europa and Ganymede from aperture photometry. A geometric model was developed to explain the light curve observed. Our results are compared with theoretical predictions (O-C). We found that our simple geometric model can explain the QSME fairly accurately, and the QSME light curve is a superposition of the light curves of an eclipse and an occultation. Notably, the observed flux drops are within 2.6% of the theoretical predictions. The size of the event central time O-Cs ranges from -14.4 to 43.2 s. Both O-Cs of flux drop and timing are comparable to other studies adopting more complicated models. Given the event rarity, model simplicity and accuracy, we encourage more observations and analysis on QSMEs to improve Solar System ephemerides.Comment: 23 pages, 5 appendixes, 16 figures, 7 table

Microfluidic processors for accurate single-cell genome sequencing

Accurate genome-scale identification of somatic mutations in single mammalian cells remains very challenging. Current single-cell genome sequencing approaches generate tens of thousands of false positive calls per genome. This is manageable for calling the millions of germline variants or single nucleotide polymorphisms (SNP).However, the number of false positives could greatly outnumber that of true somatic mutations per genome, resulting in an unacceptable level of false discovery rate and limiting the clinical application of single-cell genome sequencing. In this dissertation, I describe a strategy called SISSOR (SIngle-Stranded Sequencing in micrOfluidic Reactors) to overcome this limitation. A microfluidic processor was designed to enable the separation of long single-stranded DNA strands in a single mammalian cell, random partitioning of megabase-size single-stranded molecules into multiple nanoliter-size micro-reactors for unbiased amplification, and sequencing library construction. By separating, amplifying and sequencing megabase-size Watson and Crick DNA strands of the homologous chromosome pairs in a single cell, potential errors due to amplification and sequencing can be removed using the consensus calls on the two complementary strands, and long-range haplotype assembly can also be obtained. Using the microfluidic processor, I implemented the strategy by actual amplification and sequencing of the genomes for three single cells. I demonstrated that sequencing accuracy can be improved by two orders of magnitude and the length of haplotype assembly can also be dramatically increased as compared to other currently available methods

Expansion of human pluripotent stem cells with synthetic oolymer PMVE-alt-MA

The differentiation potential of human pluripotent stem cells (hPSCs) promises to treat degenerative diseases with cell replacement therapies. Current culture media and substrates are not only expensive, but are undefined for clinical use because of the various known and unknown animal components that are required to promote proliferation and differentiation. Chemically defined materials are ideal substitutes for hPSC culture. This study focused on the use of PMVE-alt-MA, a synthetic polymer that was previously identified by arrayed screening technology for ex vivo hPSCs maintenance. Synthetic hydrogels and microcarriers endowed with PMVE- alt-MA moieties were used to evaluate hPSC expansion, adhesion, proliferation, colony formation and maintenance. Flow cytometry and realtime PCR were used to characterize the expression of pluripotency markers in both hydrogel and microcarrier cultures. Protein adsorption to PMVE-alt- MA was found necessary for initial cell adhesion. Enzymatic dissociation with Accutase TM was optimized to passage the embryonic stem cell line Hues9 on 9.7% semi- IPN hydrogel for 8 passages and the result was comparable to Matrigel culture. However, polymer coatings on polyacrylamide hydrogel and microcarriers were found to be insufficient for stem cell self-renewal and attachment respectively. Embryoid bodies (EBs) with uniform size and shape were observed in microcarrier suspension but no difference was found in the differentiation pattern when compared to normal suspension EB formation. Our experimental results demonstrated the potential to use PMVE-alt-MA in hPSC expansion, as expected from the arrayed screening technolog

In vitro effects of arsenic trioxide on head and neck squamous cells carcinoma

abstractpublished_or_final_versionMedicineMasterMaster of Philosoph

Ultraaccurate genome sequencing and haplotyping of single human cells.

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs