Methods in Brief

Here is a selection of recently published methods papers curated by Nature Methods editors.
Published in Protocols & Methods

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Every month, Nature Methods editors meet and discuss recently published method-y papers that beyond Nature titles. We then select the papers that interest a broad audience and cover these papers on the journal page as Research Highlights. Yet, we cannot highlight all the exciting method papers in the Research Highlight section. In this post, we present the highlight candidates that we have discussed in the editorial meeting.


Garg et al. Chromosome-scale, haplotype-resolved assembly of human genomes. Nat Biotechnol (2020).
Porubsky et al. Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads. Nat Biotechnol (2020).
Di Genova et al. Efficient hybrid de novo assembly of human genomes with WENGAN. Nat Biotechnol (2020).
The arsenal of genome sequencing technologies and computational tools is expanding fast. Garg et al., Porubsky et al. and Di Genova et al. leveraged combinations of different sequencing methods and algorithmic innovations, to generate more complete/phased human genomes.

Varabyou et al. Effects of transcriptional noise on estimates of gene and transcript expression in RNA sequencing experiments. Genome Res DOI: 10.1101/gr.266213.120 (2020).
Assembly and quantification of genes and transcripts using RNA-seq data is one of the most common tasks in omics data analysis. Simulated data is often used to evaluate computational methods for these analyses. Varabyou et al incorporated transcriptional noise into simulations, and found it negatively impacts the performance of such tools.


Strickfaden et al. Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells. Cell 183, 1772–1784 (2020). 
This paper studied the physical properties of chromatin in vitro and in vivo, which suggests chromatin can function as solid-like material. The solid properties generate an elastic gel and serve as scaffold for liquid-liquid phase separation of chromatin proteins.

Subramanian, K. et al. Optical plasticity of mammalian cells. Journal of Biophysics. (2020).
Researchers have shown that cultured Chinese Hamster Ovary (CHO) cells can be evolved to greatly increase their transparency.  The cell lines with increased transparency had altered transcriptomic profiles and reduced nuclear granularity. This work could lay the groundwork for genetic tissue clearing.


Onischenko E, et al. Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling. Cell, DOI: 10.1016/j.cell.2020.11.001. 
This paper describes kinetic analysis of incorporation rates in macromolecular assemblies (KARMA), a method to analyze the native assembly kinetics of protein complexes. It makes use of affinity tagging the different components of a protein complex, pulse labeling by SILAC, and DIA mass spectrometry to compute the extent of isotopic protein labeling for each component. KARMA is used to study the co-assembly and maturation kinetics of 320 pairs of nucleoporins that form the nuclear pore complex (NPC) in yeast.

Kuljanin M, et al. Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. Nat Biotechnol (2021). 
This paper describes cysteine activity-based protein profiling (SLC-ABPP), a method to profile reactive cysteine sites. This is suitable for screening large compound libraries in a high throughput manner and boasts of reduced instrument time, reduced sample requirements, and sample multiplexing. They use the approach to identify proteome-wide targets of covalent inhibitors to mutant KRAS and Bruton’s tyrosine kinase (BTK). They also report a resource of cysteine reactivity to 285 electrophiles in three human cell lines.


Vo, P.L.H., et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol (2020).
This paper describes a streamlined INTEGRATE system that fuses Tn7-like transposes with CRISPR targeting to achieve DNA integration of up to 10 kb in bacteria. Comparing to the previous three-plasmid expression system, the single-plasmid system yields increased efficiency of integration while maintaining on-target specificity. The improved INTEGRATE also allows multiplexed insertions by using a multi-spacer CRISPR arrays.


Stickels, R.R. et al. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seqV2. Nat Biotechnol (2020).
This paper presents the version 2 of Slide-seq that has near-single-cell resolution and reaches RNA capture efficiency of 50% that is ~-10-fold greater than the original version of Slide-seq. Slide-seqV2 improves library generation, bead synthesis and array indexing, which is applied in the embryonic mouse neocortex for studying gene expression changes in development. 

Chen, W. et al. A multicenter study benchmarking single-cell RNA sequencing technologies using reference samples. Nat Biotechnol (2020). 
This paper presents a benchmarking study that compares scRNA-seq platforms and data processing from multiple centers using two reference cell samples. Interestingly, they found batch-effect correction was the most important factor in cell classification. In additional, cellular heterogeneity and platform are important when considering which bioinformatics methods should be used.


Linghu, C. et al. Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics. Cell 183, 1682–1698 (2020).
Fluorescent sensors of important signaling molecules like cAMP, PKA, and calcium were multiplexed by sending them to ‘signaling reporter islands’ (SiRIs) within cells using modularly designed clustering molecules. SiRIs were shown to work for multiplexed sensing in cultured neurons and brain slices.

Høgset, H. et al. In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy. Nat. Commun. 11: 6172 (2020). 
Label free confocal Raman microscopy enables detailed imaging of the intact zebrafish embryo. The approach was used to map subcellular details, distinguish wild-type and mutant infections in a zebrafish model of tuberculosis, and for longitudinal monitoring of wound healing.

Fang, C. et al. Minutes-timescale 3D isotropic imaging of entire organs at subcellular resolution by content-aware compressed-sensing light-sheet microscopy. Nat. Commun. 12:107 (2021).
A computational light sheet fluorescence microscopy combines dual-side confocally-scanned Bessel light-sheet illumination for deep tissue imaging with a content-aware compressed sensing algorithmic workflow to improve image quality of large samples. The approach enables fast and high quality images of structures such as the whole mouse brain with subcellular resolution.

Mazidi, H. et al. Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy. Nat. Commun. 11: 6353 (2020).
Wasserstein-induced flux (WIF) can be used to measure the confidence of individual localizations in single-molecule localization microscopy without ground truth knowledge of the sample. It can be used to measure the accuracy of reconstructions on 2D and 3D data and can be used to identify hidden molecular heterogeneities.

Qian, Y. et al. Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging. PLoS Biol 18: e3000965.
Two improved variants of the near infrared calcium sensor NIR-GECO1, NIR-GECO2 and NIR-GECO2G show improved performance in cultured cells abd mouse brain slices, and in vivo in C. elegans and X. laevis.


Viant, C. et al. Antibody Affinity Shapes the Choice between Memory and Germinal Center B Cell Fates. Cell 183(5): 1298-1311 (2020).
Viant et al have developed an unbiased fate mapping approach to analyze the trajectory of B cell fates in the context of antibody affinity. The researchers develop a mouse model where they use S1pr2-expressing mice tagged with FPs. S1pr2 is expressed in pre-germinal center (GC) or GC B cells but not naïve B cells. Then the mice are immunized and tamoxifen treatment labels cells with either ZSGreen or TdTomato (GC or memory B cells) but not follicular B cells. They show that antibody affinity governs the choice between memory or GC B cells.


Yemini, E. et al. NeuroPAL: A Multicolor Atlas for Whole-Brain Neuronal Identification in C. elegans. Cell 184:272-288 (2021).
NeuroPAL is a transgene consisting of 41 reporters that allows unambiguous identification of all C. elegans neurons based on their color combination and position. As the NeuroPAL color spectrum does not overlap with the emission from GFP-based tools, NeuroPAL can readily be combined with popular tools such as for example GCaMP. Yemini et al. use NeuroPAL for co-expression studies with reporters for GABA and other neurotransmitter receptor expression.

Unger, E.K. et al. Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning. Cell 183:1986-2002 (2020).
iSeroSnFR is a serotonin sensor derived from a choline-binding microbial periplasmic binding protein, whose binding pocket was redesigned using machine learning. After characterization in cell culture experiments, the researchers demonstrated the iSeroSnFR’s capabilities in mouse brain slices, or in vivo using fiber photometry.


Nishimura, T. et al. Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras. Cell Stem Cell 28(1):141-149 (2020).
Nishimura et al create a cell competitive niche in mouse embryos to improve intra and inter-species chimeras. They deleted Insulin-like growth factor 1 (Igf1) using CRISPR in early stages of development. They show that the Igf1null mice had a significantly high connective tissue: blood and organ:blood chimeric ratio (from donor mice) up to E18.5.  This approach could also be applied to create mice-rat chimeras. 

Arai, F. et al. Machine Learning of Hematopoietic Stem Cell Divisions from Paired Daughter Cell Expression Profiles Reveals Effects of Aging on Self-Renewal. Cell Systems 11(6): 640-652 (2020).
In this work, the researchers trained an ANN to identify cells in the HSCs division pathway and predict their age from their gene expression pattern. They found that the age of a HSC controlled its ability to self-renew. They also found that niche factors like ANGPT1 etc. have the ability to modulate division patterns which can vary with age.

McDonald, D. et al. Defining the Teratoma as a Model for Multi-lineage Human Development. Cell 183(5):1402-1419 (2020).
McDonald et al propose the use of a teratoma for validating pluripotency in stem cells. They perform scRNAseq of ~180k cells from 23 teratomas and found that the cells recap upto 20 cell types across all three germ layers.  CRISPR screens show that the teratomas could be used to study the effects of genetic perturbations.

Wang, H.F. et al. Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen. Cell Reports 33(4):108309 (2020).
Wang et al develop FOCUS (features-oriented CRISPR-utilized systematic), a screen for cis regulatory elements (CREs). They analyzed the CREs for OCT4 (important pluripotency marker) with the aim of identifying functional enhancers for pluripotency maintenance in embryonic stem cells (ESC). The researchers collected ATAC-seq data for ESCs to determine the accessible regions. Then sgRNAs targeting accessible OCT4 CREs were transfected into the ESCs for functional genetic screens.

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