Reliable and accurate diagnostics from highly multiplexed sequencing assays

Scalable, inexpensive, and secure testing for SARS-CoV-2 infection is crucial for control of the novel coronavirus pandemic. Recently developed highly multiplexed sequencing assays (HMSAs) that rely on high-throughput sequencing can, in principle, meet these demands, and present promising alternatives to currently used RT-qPCR-based tests. However, reliable analysis, interpretation, and clinical use of HMSAs requires overcoming several computational, statistical and engineering challenges. Using recently acquired experimental data, we present and validate a computational workflow based on kallisto and bustools, that utilizes robust statistical methods and fast, memory efficient algorithms, to quickly, accurately and reliably process high-throughput sequencing data. We show that our workflow is effective at processing data from all recently proposed SARS-CoV-2 sequencing based diagnostic tests, and is generally applicable to any diagnostic HMSA

Highly multiplexed single-cell RNA-seq by DNA oligonucleotide tagging of cellular proteins

We describe a universal sample multiplexing method for single-cell RNA sequencing in which fixed cells are chemically labeled by attaching identifying DNA oligonucleotides to cellular proteins. Analysis of a 96-plex perturbation experiment revealed changes in cell population structure and transcriptional states that cannot be discerned from bulk measurements, establishing an efficient method for surveying cell populations from large experiments or clinical samples with the depth and resolution of single-cell RNA sequencing

Highly multiplexed single-cell RNA-seq by DNA oligonucleotide tagging of cellular proteins

We describe a universal sample multiplexing method for single-cell RNA sequencing in which fixed cells are chemically labeled by attaching identifying DNA oligonucleotides to cellular proteins. Analysis of a 96-plex perturbation experiment revealed changes in cell population structure and transcriptional states that cannot be discerned from bulk measurements, establishing an efficient method for surveying cell populations from large experiments or clinical samples with the depth and resolution of single-cell RNA sequencing

Principles of open source bioinstrumentation applied to the poseidon syringe pump system

The poseidon syringe pump and microscope system is an open source alternative to commercial systems. It costs less than $400 and can be assembled in under an hour using the instructions and source files available at https://pachterlab.github.io/poseidon. We describe the poseidon system and use it to illustrate design principles that can facilitate the adoption and development of open source bioinstruments. The principles are functionality, robustness, safety, simplicity, modularity, benchmarking, and documentation

Reliable and accurate diagnostics from highly multiplexed sequencing assays

Scalable, inexpensive, and secure testing for SARS-CoV-2 infection is crucial for control of the novel coronavirus pandemic. Recently developed highly multiplexed sequencing assays (HMSAs) that rely on high-throughput sequencing can, in principle, meet these demands, and present promising alternatives to currently used RT-qPCR-based tests. However, reliable analysis, interpretation, and clinical use of HMSAs requires overcoming several computational, statistical and engineering challenges. Using recently acquired experimental data, we present and validate a computational workflow based on kallisto and bustools, that utilizes robust statistical methods and fast, memory efficient algorithms, to quickly, accurately and reliably process high-throughput sequencing data. We show that our workflow is effective at processing data from all recently proposed SARS-CoV-2 sequencing based diagnostic tests, and is generally applicable to any diagnostic HMSA

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme–substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems

Modular and efficient pre-processing of single-cell RNA-seq

Analysis of single-cell RNA-seq data begins with the pre-processing of reads to generate count matrices. We investigate algorithm choices for the challenges of pre-processing, and describe a workflow that balances efficiency and accuracy. Our workflow is based on the kallisto and bustools programs, and is near-optimal in speed and memory. The workflow is modular, and we demonstrate its flexibility by showing how it can be used for RNA velocity analyses