The Nature Methods editorial team gets together each month to pick interesting new methods developments that are published in other journals for the Research Highlights section. Unfortunately, we can't highlight all the great methods being developed. Below are a few exciting papers that we couldn't feature in our October issue.

BIOCHEMISTRY AND PROTEOMICS

Trussart, M. et al. Removing unwanted variation with CytofRUV to integrate multiple CyTOF datasets. Elife. 9, e59630 (2020).

CyTOF, or mass cytometry, studies can suffer from batch effects, which can make it difficult to integrate datasets taken on different days or in different locations. Trussart et al. develop CyroRUV, which uses Remove Unwanted Variation (RUV) to analyze multiple batches, correct for batch effects, and integrate data. CytoRUV is implemented as an R-Shinny application.

Ma, G. et al. Optical imaging of single-protein size, charge, mobility, and binding. Nat. Commun. 11, 4768 (2020). 

An optical method is described to determine the charge, size, and mobility of single-proteins. This method begins by tethering a protein of interest to a surface with a flexible polymer. The tethered protein is then driven into oscillation with an electric field. This oscillation reveals the size, charge, and mobility features of the protein and is imaged with near field optical imaging.

Xu, C. et al. Computational design of transmembrane pores. Nature 585, 129–134 (2020).  

The paper describes a Rosetta based design strategy for the computational design of transmembrane pores using two concentric rings of alpha-helices. First, a water-soluble pore containing structure is designed, followed by its conversion to a transmembrane pore with strategic amino acid mutations. X-ray crystallography and cryo-EM was used to determine the structures of a 12-helical and a 16-helical pore, respectively. Patch clamp experiments in insect cells showed that ions can pass through the designed 12-helical pore, and that the pore was more selective for potassium over sodium. The 16-helical pore was incorporated in liposomes and enabled the passage of biotinylated Alexa Fluor 488, a green fluorescent dye.

Jiang, L. et al. "A quantitative proteome map of the human body." Cell 183, 269-283 (2020).  

This resource quantifies relative protein levels from 12,000 genes across 32 normal human tissues. Tissue specific protein and proteins enriched in specific tissues were identified and compared to transcriptome data. Differences between RNA and protein enrichment levels in some key cases suggest a more complex regulation or protein secretion patterns.

CELL BIOLOGY

Wang et al. Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle. Nat. Medicine https://doi.org/10.1038/s41591-020-1040-z (2020).

Wang et al perform a longitudinal single cell transcriptomic analysis of the human endometrium during the human menstrual cycle. They discover gene signatures for each cell type across the four phases of endometrial transformation.

GENOMICS AND GENETICS

Kim, H.K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. (2020). https://doi.org/10.1038/s41587-020-0677-y

Prime editing with its versatile ability to introduce different types of editing has become popular. In this work, Kim et al studied the factors that affect the editing efficiency of Prime Editor 2, which include the pegRNA design and editing context. Unlike standard guide RNA design, pegRNA used in prime editing requires an additional extension for reverse transcription (RT), including an RT template and a primer binding site that serves as the point of the initiation for RT. This paper provides practical guides for pegRNA design and computational models for predicting editing outcomes.

Jones, S.K. et al. Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nat. Biotechnol. (2020). https://doi.org/10.1038/s41587-020-0646-5

Binding affinity and cleavage kinetics are important biochemical properties to understand enzyme fidelity. To measure the cleavage kinetics of WT and engineered Cas nucleases, Jones et al describes NucleaSeq that sequences time-resolved cleavage products of guide RNA-matched and mispaired DNA targets. The researchers also integrate CHAMP assay into NucleaSeq to profile the binding profiles of Cas9 variants and Cas12a. The characterized biochemical properties will help users to better select nucleases for different editing applications, as well as help developers to improve computational tools that are designed to predict editing outcomes. 

Nurk et al. HiCanu: accurate assembly of segmental duplications, satellites, and allelic variants from high-fidelity long reads. Genome Res. 30, 1291-1305 (2020).

PacBio HiFi is a recent sequencing technology that generates accurate long reads. Nurk et al develop HiCanu, a modification of the Canu assembler, that can resolve difficult-to-assemble genomic regions such as segmental duplications, major histocompatibility complex variants, satellite DNAs, and capture both alleles.

Martiniano et al. Removing reference bias and improving indel calling in ancient DNA data analysis by mapping to a sequence variation graph. Genome Biol. 21: 250 (2020).

Sequencing of ancient DNA (aDNA) can uncover rich information about population history and evolution. However, substantial degradation and contamination pose major challenges to its analysis. Martiniano et al apply the variation graph (vg) to align aDNA data with reduced reference bias and more sensitive variant calling especially for indels.

IMAGING AND MICROSCOPY

Tamura, T. et al. Organelle membrane-specific chemical labeling and dynamic imaging in living cells. Nat Chem. Biol. (2020). https://doi.org/10.1038/s41589-020-00651-z

Choline-containing phospholipids (CPLs), including phosphatidylcholine (PC) and sphingomyelin (SM) are crucial for proper membrane function in eukaryotes. Tamura et al. present a method for selective fluorescent labeling of CPLs in the ER-Golgi network or mitochondria. The method works by metabolic incorporation of azido-choline into CPLs in the cell membrane followed by spatially limited strain-promoted alkyne–azide cycloaddition (SPAAC) of organelle-localizable click reagents (OCRs) bearing a strained alkyne. The probes were used to study interorganelle trafficking of these CPLs.

Li, J.H. et al. Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles. Nat Commun 11, 4259 (2020).

Li et al. present a versatile strategy for tracking and manipulating membrane proteins. They use fluorescent magnetic particles to track membrane proteins at the single-molecule level with high spatiotemporal resolution. They also use a magnetic needle to pull membrane components, which allows them to detect barriers to diffusion like the cytoskeleton.

IMMUNOLOGY

Barennes et al. Benchmarking of T cell receptor repertoire profiling methods reveals large systematic biases. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0656-3 (2020).

Barennes et al perform a systematic analysis of nine TCR sequencing methods to find large differences in accuracy and reproducibility for TCR alpha and TCR beta chains. They conclude that methods are either optimized to capture clonal diversity or to identify rare clonotypes.

Corridoni et al Single-cell atlas of colonic CD8+ T cells in ulcerative colitis. Nature Medicine 26, 1480–1490 (2020)

Using single cell transcriptomics, Corridoni et al have developed an atlas of colonic CD*+ T cells in ulcerative colitis and healthy controls. Extensive analysis shows the presence of IL26+ dysfunctional CD8+ T cells in the colon  like contributing to the inflammatory microenvironment in ulcerative colitis.

NEUROSCIENCE

Scheffer, L.K. et al. A connectome and analysis of the adult Drosophila central brain. Elife 9: e57443 (2020).

Using FIB-SEM, Scheffer et al. have obtained the full connectome of the adult Drosophila central brain consisting of about 25,000 neurons and 20 million synapses. To cope with the wealth of data, automated segmentation and synapse prediction methods were used in the reconstruction. Furthermore, the researchers annotated cell types and present additional analyses. The data can be accessed at https://neuprint.janelia.org/.

Suzuki, K. et al. A synthetic synaptic organizer protein restores glutamatergic neuronal circuits. Science 369: eabb4853 (2020).

Suzuki et al. developed a synthetic synaptic organizer complex. Consisting of domains of the extracellular scaffolding proteins cerebellin-1 and pentraxin-1, this complex instructs the formation of pre- and post-synaptic structures when injected into neural tissue. In mouse models, injection of the complex ameliorated deficits in ataxia, Alzheimer’s disease and spinal cord injury.

Drokhlyansky, E. et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell 182, 1606-1622 (2020).

The enteric nervous system has been challenging to characterize with single-cell or single-nucleus transcriptomic approaches. Drokhlyansky et al. have modified existing protocols and come up with RAISIN RNA-seq to analyze single intact nuclei with associated rough endoplasmic reticulum. To extend the approach to the human enteric nervous system, the researchers came up with MIRACL-seq, which enriches for rare cell types. Using these methods, the researchers profiled 1.5 million nuclei from the mouse ileum and colon, and the human colon.