Highly Parallel Profiling of Cas9 Variant Specificity.

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

A comparison of Oxford nanopore library strategies for bacterial genomics.

BACKGROUND: Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT's recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. RESULTS: Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. CONCLUSION: The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads.

T-RHEX-RNAseq - a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

BACKGROUND: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging. RESULTS: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. CONCLUSION: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

Transposase-assisted tagmentation: an economical and scalable strategy for single-worm whole-genome sequencing.

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

SPO-Seq: An Accessible Method for Efficient Evaluation of Spo11 Catalytic Activity and Profiling Meiotic DSB Hotspots in Saccharomyces cerevisiae.

Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.

Hi-Tag: a simple and efficient method for identifying protein-mediated long-range chromatin interactions with low cell numbers.

Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation. However, these techniques require complex experimental procedures and millions of cells per experiment, which limits their widespread application in life science research. Here, we develop a novel method, Hi-Tag, that identifies high-resolution, long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation (with a phosphorothioate-modified linker). Hi-Tag can be implemented using as few as 100,000 cells, involving simple experimental procedures that can be completed within 1.5 days. Meanwhile, Hi-Tag is capable of using its own data to identify the binding sites of specific proteins, based on which, it can acquire accurate interaction information. Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies, particularly in the context of limited cell availability.

Cut&tag: a powerful epigenetic tool for chromatin profiling.

Analysis of transcription factors and chromatin modifications at the genome-wide level provides insights into gene regulatory processes, such as transcription, cell differentiation and cellular response. Chromatin immunoprecipitation is the most popular and powerful approach for mapping chromatin, and other enzyme-tethering techniques have recently become available for living cells. Among these, Cleavage Under Targets and Tagmentation (CUT&Tag) is a relatively novel chromatin profiling method that has rapidly gained popularity in the field of epigenetics since 2019. It has also been widely adapted to map chromatin modifications and TFs in different species, illustrating the association of these chromatin epitopes with various physiological and pathological processes. Scalable single-cell CUT&Tag can be combined with distinct platforms to distinguish cellular identity, epigenetic features and even spatial chromatin profiling. In addition, CUT&Tag has been developed as a strategy for joint profiling of the epigenome, transcriptome or proteome on the same sample. In this review, we will mainly consolidate the applications of CUT&Tag and its derivatives on different platforms, give a detailed explanation of the pros and cons of this technique as well as the potential development trends and applications in the future.

Integrative Analysis of CUT&Tag and RNA-Seq Data Through Bioinformatics: A Unified Workflow for Enhanced Insights.

Cleavage Under Targets and Tagmentation (CUT&Tag) is a recent methodology used for robust epigenomic profiling that, unlike conventional chromatin immunoprecipitation (ChIP-Seq), requires only a limited amount of cells as starting material. RNA sequencing (RNA-Seq) reveals the presence and quantity of RNA in a biological sample, describing the continuously changing cellular transcriptome. The integrated analysis of transcriptional activity, histone modifications, and chromatin accessibility via CUT&Tag is still in its infancy compared to the well-established ChIP-Seq. This chapter describes a robust bioinformatics methodology and workflow to perform an integrative CUT&Tag/RNA-Seq analysis.

Tn5 DNA Transposase in Multi-Omics Research.

Tn5 transposase use in biotechnology has substantially advanced the sequencing applications of genome-wide analysis of cells. This is mainly due to the ability of Tn5 transposase to efficiently transpose DNA essentially randomly into any target DNA without the aid of other factors. This concise review is focused on the advances in Tn5 applications in multi-omics technologies, genome-wide profiling, and Tn5 hybrid molecule creation. The possibilities of other transposase uses are also discussed.

Analyzing the Genome-Wide Distribution of Histone Marks by CUT&Tag in Drosophila Embryos.

CUT&Tag is a method to map the genome-wide distribution of histone modifications and some chromatin-associated proteins. CUT&Tag relies on antibody-targeted chromatin tagmentation and can easily be scaled up or automatized. This protocol provides clear experimental guidelines and helpful considerations when planning and executing CUT&Tag experiments.

Selection of Flax Genotypes for Pan-Genomic Studies by Sequencing Tagmentation-Based Transcriptome Libraries.

Flax (Linum usitatissimum L.) products are used in the food, pharmaceutical, textile, polymer, medical, and other industries. The creation of a pan-genome will be an important advance in flax research and breeding. The selection of flax genotypes that sufficiently cover the species diversity is a crucial step for the pan-genomic study. For this purpose, we have adapted a method based on Illumina sequencing of transcriptome libraries prepared using the Tn5 transposase (tagmentase). This approach reduces the cost of sample preparation compared to commercial kits and allows the generation of a large number of cDNA libraries in a short time. RNA-seq data were obtained for 192 flax plants (3-6 individual plants from 44 flax accessions of different morphology and geographical origin). Evaluation of the genetic relationship between flax plants based on the sequencing data revealed incorrect species identification for five accessions. Therefore, these accessions were excluded from the sample set for the pan-genomic study. For the remaining samples, typical genotypes were selected to provide the most comprehensive genetic diversity of flax for pan-genome construction. Thus, high-throughput sequencing of tagmentation-based transcriptome libraries showed high efficiency in assessing the genetic relationship of flax samples and allowed us to select genotypes for the flax pan-genomic analysis.

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

Integration site (IS) analysis is essential in ensuring safety and efficacy of gene therapies when integrating vectors are used. Although clinical trials of gene therapy are rapidly increasing, current methods have limited use in clinical settings because of their lengthy protocols. Here, we describe a novel genome-wide IS analysis method, "detection of the integration sites in a time-efficient manner, quantifying clonal size using tagmentation sequencing" (DIStinct-seq). In DIStinct-seq, a bead-linked Tn5 transposome is used, allowing the sequencing library to be prepared within a single day. We validated the quantification performance of DIStinct-seq for measuring clonal size with clones of known IS. Using ex vivo chimeric antigen receptor (CAR)-T cells, we revealed the characteristics of lentiviral IS. We then applied it to CAR-T cells collected at various times from tumor-engrafted mice, detecting 1,034-6,233 IS. Notably, we observed that the highly expanded clones had a higher integration frequency in the transcription units and vice versa in genomic safe harbors (GSH). Also, in GSH, persistent clones had more frequent IS. Together with these findings, the new IS analysis method will help to improve the safety and efficacy of gene therapies.

Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells.

Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to other organisms.

MuA-based Molecular Indexing for Rare Mutation Detection by Next-Generation Sequencing.

Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases. Here we demonstrate the remarkable tolerance of MuA transposase to the presence of multiple replacements in transposon sequence, and explore this unique feature to engineer the MuA transposome complex with randomised nucleotides in 12 transposon positions, which can be introduced as a barcode into the target molecule after transposition event. We applied the approach of Unique MuA-based Molecular Indexing (UMAMI) to assess the power of rare mutation detection by shortgun sequencing on the Illumina platform. Our results show that UMAMI allows detection of rare mutations readily and reliably, and in this paper we report error rate values for the number of thermophilic DNA polymerases measured by using UMAMI.

Simultaneous Tagmentation-Based Detection of ChIP/ATAC Signal with Bisulfite Sequencing.

DNA methylation is thought to regulate accessibility of chromatin and binding of regulatory elements; however, it is difficult to determine if chromatin accessibility or transcription factor (TF) binding overlap with methylated or unmethylated DNA if the assays are performed separately. In order to examine accessibility or TF binding simultaneously with methylation on the same DNA molecule, we developed EpiMethylTag which combines ATAC-Seq or ChIP-Seq (M-ATAC or M-ChIP) with bisulfite conversion. Our approach provides a fast, low-input, low sequencing depth method to determine whether DNAme and accessibility/TF binding are mutually exclusive or can coexist in certain locations.

Concurrent mapping of multiple epigenetic marks and co-occupancy using ACT2-seq.

BACKGROUND: Genome-wide profiling of epigenetic marks is a core technology in molecular genetics. Co-occupancy of different epigenetic marks or protein factors at the same genomic locations must often be inferred from multiple independently collected data sets. However, this strategy does not provide direct evidence of co-enrichment in the same cells due to the existence of cellular heterogeneity. To address this issue, we have developed a technique termed ACT2-seq that is capable of concurrently profiling multiple epigenetic marks in a single biological sample. In addition to reducing the numbers of samples required for experiments, ACT2-seq is capable of mapping co-occupancy of epigenetic factors on chromatin. This strategy provides direct evidence of co-enrichment without requiring complex single-molecule, single-cell, or magnetic bead-based approaches. RESULTS: We concurrently profiled pairs of two epigenetic marks using ACT2-seq as well as three marks in individual samples. Data obtained using ACT2-seq were found to be reproducible and robust. ACT2-seq was capable of cleanly partitioning concurrently mapped data sets that exhibited distinct enrichment patterns. Using ACT2-seq, we identified distinct relationships between co-occupancy of specific histone modifications and gene expression patterns. CONCLUSIONS: We conclude that ACT2-seq presents an attractive option for epigenomic profiling due to its ease of use, potential for reducing sample and sequencing costs, and ability to simultaneously profile co-occupancy of multiple histone marks and/or chromatin-associated proteins.

Colorimetric RT-LAMP and LAMP-sequencing forDetecting SARS-CoV-2 RNA in Clinical Samples.

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi et al., 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.

Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue.

Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.

ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach.

Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis.

tagHi-C Reveals 3D Chromatin Architecture Dynamics during Mouse Hematopoiesis.

Spatiotemporal chromatin reorganization during hematopoietic differentiation has not been comprehensively characterized, mainly because of the large numbers of starting cells required for current chromatin conformation capture approaches. Here, we introduce a low-input tagmentation-based Hi-C (tagHi-C) method to capture the chromatin structures of hundreds of cells. Using tagHi-C, we are able to map the spatiotemporal dynamics of chromatin structure in ten primary hematopoietic stem, progenitor, and differentiated cell populations from mouse bone marrow. Our results reveal that changes in compartment dynamics and the Rabl configuration occur during hematopoietic cell differentiation. We identify gene-body-associating domains (GADs) as general structures for highly expressed genes. Moreover, we extend the body of knowledge regarding genes influenced by genome-wide association study (GWAS) loci through spatial chromatin looping. Our study provides the tagHi-C method for studying the three-dimensional (3D) genome of a small number of cells and maps the comprehensive 3D chromatin landscape of bone marrow hematopoietic cells.