Biology subjects

May-Zhang, A.

Publications and source records attributed to May-Zhang, A..

2 recordsLinked to original sources

bioRxiv2024

Intrinsic molecular identifiers enable robust molecular counting in single-cell sequencing

Particle-templated instant partition sequencing (PIPseq), is an emerging approach for massively scalable single-cell gene expression studies that does not require complex instrumentation or expensive consumables. We present PIPseqTM V, a novel implementation of the PIPseq workflow with significant improvements in assay performance and sensitivity compared to prior methods. Among the innovations driving the improved performance in PIPseq V is a new approach for transcript counting using Intrinsic Molecular Identifiers (IMIs) from the captured transcript sequence, eliminating the need for traditional Unique Molecular Identifiers (UMIs). IMIs are the starting positions for molecules generated by random fragmentation after limited cycle PCR, which can be used to uniquely identify individual transcript copies of genes expressed within individual cells. To correct for experimental variation in IMI generation, we have developed a dynamic correction strategy that can adapt to different sample cell input, transcript expression, and sequencing depth without the need for external benchmarking or controls. Our results demonstrate that PIPseq V with IMI-based analysis provides biological information comparable to established UMI-based approaches while avoiding UMI-associated biases. Dynamic correction offers a robust and data-driven analysis strategy for scRNAseq.

genomics↗

bioRxiv2022

Microfluidics-free single-cell genomics with templated emulsification

Single-cell RNA sequencing is now a standard method used to reveal the molecular details of cellular heterogeneity, but current approaches have limitations on speed, scale, and ease of use that stem from the complex microfluidic devices or fluid handling steps required for sample processing. We, therefore, developed a method that does not require specialized microfluidic devices, expertise, or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. PIP-seq accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multi-omics measurements, and can accurately characterize cell types in human breast tissue when compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq revealed the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible, and scalable next-generation workflow that extends single-cell sequencing to new applications, including screening, diagnostics, and disease monitoring.

genomics↗