Linfeng Xu

Recent developments in PacBio high-fidelity (HiFi) sequencing technologies have transformed genomic research, with circular consensus sequencing now achieving 99.9% accuracy for long (up to 25 kb) single-molecule reads. This method circumvents biases intrinsic to amplification-based approaches, enabling thorough analysis of complex genomic regions [including tandem repeats, segmental duplications, ribosomal DNA (rDNA) arrays, and centromeres] as well as direct detection of base modifications, furnishing both sequence and epigenetic data concurrently. This has streamlined a number of tasks including genome assembly, variant detection, and full-length transcript analysis. This review provides a comprehensive overview of the applications and challenges of HiFi sequencing across various fields, including genomics, transcriptomics, and epigenetics. By delineating the evolving landscape of HiFi sequencing in multi-omics research, we highlight its potential to deepen our understanding of genetic mechanisms and to advance precision medicine.

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq (Easily Accessible Single microbe sequencing). By adapting the single cell workflow of the commercial Mission Bio Tapestri instrument, this method allows for efficient sequencing of individual microbes’ genomes. EASi-seq allows thousands of microbes to be sequenced per run and, as we show, can generate detailed atlases of human and environmental microbiomes. The ability to capture large shotgun genome datasets from thousands of single microbes provides new opportunities in discovering and analyzing species subpopulations. To facilitate this, we develop a companion bioinformatic pipeline that clusters genome by sequence similarity, improving whole genome assembly, strain identification, taxonomic classification, and gene annotation. In addition, we demonstrate integration of metagenomic contigs with the EASi-seq datasets to reduce capture bias and increase coverage. Overall, EASi-seq enables high quality single cell genomic data for microbiome samples using an accessible workflow that can be run on a commercially available platform.

Significance: The study of specific cell–cell interactions at scale would be a significant advancement in single-cell biology with clear utility in immuno-oncology. Our development of Droplet Assembly provides a tool for such studies by extending the benefits of single-cell droplet microfluidics to high-order cell analyses. This technology allows for the construction, sorting, and downstream processing of cell–cell interactions and is compatible with single-cell genomic readouts.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer‐scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell‐based assays.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, we describe a simple method to fabricate substrates patterned with micron-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces. The wells can also concentrate and localize cells and beads for cell-based assays.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

Suitable micropumping methods for flow control represent a major technical hurdle in the development of microfluidic systems for point-of-care testing (POCT). Passive micropumping for point-of-care microfluidic systems provides a promising solution to such challenges, in particular, passive micropumping based on capillary force and air transfer based on the air solubility and air permeability of specific materials. There have been numerous developments and applications of micropumping techniques that are relevant to the use in POCT. Compared with active pumping methods such as syringe pumps or pressure pumps, where the flow rate can be well-tuned independent of the design of the microfluidic devices or the property of the liquids, most passive micropumping methods still suffer flow-control problems. For example, the flow rate may be set once the device has been made, and the properties of liquids may affect the flow rate. However, the advantages of passive micropumping, which include simplicity, ease of use, and low cost, make it the best choice for POCT. Here, we present a systematic review of different types of passive micropumping that are suitable for POCT, alongside existing applications based on passive micropumping. Future trends in passive micropumping are also discussed.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

We report a simple method to fabricate PDMS (polydimethylsiloxane) microwell arrays on glass by using a PDMS stamp to study cell-to-cell adhesion. In the cell-to-cell study, a glass substrate is required since glass has better cell attachment. The microwell arrays are replicated from an SU-8 master mold, and then are transferred to a glass substrate by lifting the PDMS stamp, followed by oxygen plasma bonding of the PDMS stamp on the glass substrate. For the cell-to-cell adhesion, four different types of PDMS arrays (e.g., rectangle, bowtie, wide-rhombus, and rhombus) were designed to vary the cell-to-cell contact length. The transfer success rates of the microwell arrays were measured as a function of both the contact area of the PDMS and the glass substrate and the different ratios between the base polymers and the curing agent. This method of generating the microwell arrays will enable a simple and robust construction of PDMS-based devices for various biological applications.