Accurate SNV detection in single cells by transposon-based whole-genome amplification of complementary strands.

Single-nucleotide variants (SNVs), pertinent to aging and disease, occur sporadically in the human genome, hence necessitating single-cell measurements. However, detection of single-cell SNVs suffers from false positives (FPs) due to intracellular single-stranded DNA damage and the process of whole-genome amplification (WGA). Here, we report a single-cell WGA method termed multiplexed end-tagging amplification of complementary strands (META-CS), which eliminates nearly all FPs by virtue of DNA complementarity, and achieved the highest accuracy thus far. We validated META-CS by sequencing kindred cells and human sperm, and applied it to other human tissues. Investigation of mature single human neurons revealed increasing SNVs with age and potentially unrepaired strand-specific oxidative guanine damage. We determined SNV frequencies along the genome in differentiated single human blood cells, and identified cell type-dependent mutational patterns for major types of lymphocytes.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Rapid and sensitive single-cell RNA sequencing with SHERRY2.

BACKGROUND: Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. RESULTS: Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. CONCLUSIONS: SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

Phage resistance of Salmonella enterica obtained by transposon Tn5-mediated SefR gene silent mutation.

Salmonella enterica contamination is a primary cause of global food poisoning. Using phages as bactericidal alternatives to antibiotics could confront the issue of drug resistance. However, the problem of phage resistance, especially mutant strains with multiple phage resistance, is a critical barrier to the practical application of phages. In this study, a library of EZ-Tn5 transposable mutants of susceptible host S. enterica B3-6 was constructed. After the infestation pressure of a broad-spectrum phage TP1, a mutant strain with resistance to eight phages was obtained. Analysis of the genome resequencing results revealed that the SefR gene was disrupted in the mutant strain. The mutant strain displayed a reduced adsorption rate of 42% and a significant decrease in swimming and swarming motility, as well as a significantly reduced expression of the flagellar-related FliL and FliO genes to 17% and 36%, respectively. An uninterrupted form of the SefR gene was cloned into vector pET-21a (+) and used for complementation of the mutant strain. The complemented mutant exhibited similar adsorption and motility as the wild-type control. These results suggest that the disrupted flagellar-mediated SefR gene causes an adsorption inhibition, which is responsible for the phage-resistant phenotype of the S. enterica transposition mutant.

Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei.

BACKGROUND: The "Assay for Transposase Accessible Chromatin sequencing" (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. RESULTS: In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. CONCLUSIONS: We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.

dCITI-Seq: droplet combinational indexed transposon insertion sequencing.

The rapid development of high-throughput parallel sequencing poses new challenges for large-scale barcoding and sequencing library construction. Here, we present droplet combinational indexed transposon insertion sequencing (dCITI-Seq), in which samples are indexed by the direct insertion of index-containing adaptors through transposition. The random combination of two sets of adaptors with known barcodes and massively parallel transposition was realized via a robust droplet pairing and merging platform. This strategy potentially enlarges the indexing capacity and decreases index crosstalk. Also, dCITI-Seq exhibited a lower GC base preference than conventional in-tube transposition library preparation. With a custom bioinformatic processing, it could be further applied to large-scale single-cell sequencing.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, and fliR), 4 curli fimbriae genes (csgD, csgA, csgF, and csgG) and 3 type 1 fimbriae genes (fimA, fimD, and fimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, and csgG) and other functional genes (fimC, glxK, yehE, 07045, and 11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Method for efficient soluble expression and purification of recombinant hyperactive Tn5 transposase.

Efficient preparation of libraries is the key step of next-generation sequencing (NGS) methods. Tn5 transposase enables simple, robust and highly efficient tagmentation-based library construction. Here, we report a simple and reliable expression and purification strategy based on fusing Tn5 to the small B1 immunoglobulin binding domain of Streptococcal protein G (GB1) and high affinity 10× His tag. The purified recombinant Tn5 showed high DNA tagmentation activity and ultra-low nucleic acid contamination. This method greatly cuts the costs of Tn5-based NGS library construction and is beneficial to the development of new NGS methods.

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria.

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.

Tn5 Transposase Applied in Genomics Research.

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast "cut and paste" and "copy and paste" functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.

[ATAC-seq and its applications in complex disease].

ATAC-seq is a high-throughput technology that defines and quantifies chromatin accessibility by analyzing Tn5 transposase enzymes. ATAC-seq is used to map chromatin accessibility genome-wide and to identify regions of transcription-factor binding and nucleosome position. As such, ATAC-seq is a new generation tool used in biomedical research to measure and articulate the pathogenesis of major diseases, to demonstrate the pharmacology of current drugs, and to guide the development of new drugs and the function of biomarkers. In this review, we summarize the current applications and advantages of ATAC-seq, and define its prospective contributions related to the regulatory mechanism of gene expression to identify and manage complex disease while elucidating and guiding future research references and strategies.

[Advances in assay for transposase-accessible chromatin with high-throughput sequencing].

Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) was developed in 2013. It has the advantages of more convenient operation and higher efficiency for DNA recovery than DNase I hypersensitive site sequencing (DNase-seq) and micrococcal nuclease sequencing (MNase-seq). ATAC-seq currently is the most popular technique of genome-wide mapping for chromatin accessibility. It provides information on binding regions of transcription factors and nucleosome localization on the chromatin. Thus, ATAC-seq is of great significance for studying the epigenetics and molecular mechanisms in chromatin structure. In this review, we compare the advantages and disadvantages of multiple techniques for profiling chromatin accessibility, and summarize the principles, main process, development and applications of ATAC-seq. We hope this review will provide a reference for study of genome-wide mapping for chromatin accessibility, identification of cis-regulatory elements, and dissection of the epigenetic and genetic regulatory networks using the ATAC-seq technology in eukaryotes.

Detect accessible chromatin using ATAC-sequencing, from principle to applications.

BACKGROUND: Chromatin accessibility is crucial for gene expression regulation in specific cells and in multiple biological processes. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) is an effective way to reveal chromatin accessibility at a genome-wide level. Through ATAC-seq, produced reads from a small number of cells reflect accessible regions that correspond to nucleosome positioning and transcription factor binding sites, due to probing hyperactive Tn5 transposase to DNA sequence. CONCLUSION: In this review, we summarize both principle and features of ATAC-seq, highlight its applications in basic and clinical research. ATAC-seq has generated comprehensive chromatin accessible maps, and is becoming a powerful tool to understand dynamic gene expression regulation in stem cells, early embryos and tumors.

Cytotoxic selectivity and apoptosis induction of piericidin A contributes potentially to its insecticidal effect against Mythimna separata (Lepidoptera: Noctuidae) larvae.

Piericidin A (PIA), an active inhibitor of Complex I, is widely used in studies of the anti-bacterial and anti-disease competence, but its physiological and mechanistic effects have rarely been clearly defined in insect individual or insect cells. The present study reveals the considerable insecticidal activity of PIA on Mythimna separata larvae by using a comparison with Aphis craccivora adult, and the cytotoxic selectivity induced by PIA on lepidopteran Tn5B1-4 cells. We demonstrate that the viability of Tn5B1-4 cells is inhibited by PIA in a time- and concentration-dependent manner with IC50 value of 0.061 µM, whilst PIA shows slight inhibitory effect on the viability of HepG2 and Hek293 cells with IC50 value of 233.97 and 228.96 µM, respectively. The inhibitory effect of PIA on the proliferation of Tn5B1-4 cells is significant and persistent, causing a series of morphological changes including cell shrinkage, condensed and fragmented nuclei. Intracellular biochemical assays show that PIA induces apoptosis of Tn5B1-4 cells coincides with a decrease in the mitochondrial membrane potential. PIA in Tn5B1-4 cells can be chelated by EDTA, thereby losing cytotoxicity, whereas exogenous Ca2+ restores the cytotoxicity of PIA by chelating with EDTA in a competitive manner. Our findings highlight the importance of the long-lasting cytotoxicity and the cytoxic selectivity on Tn5B1-4 cells caused by PIA, which ensure the identification of insecticidal effect of PIA against insect pests.

[Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin].

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states.

BACKGROUND: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. RESULTS: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3' overhang of 3 random nucleotides, which can be efficiently ligated to the 3' end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. CONCLUSIONS: This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

Role of Ttca of Citrobacter Werkmanii in Bacterial Growth, Biocides Resistance, Biofilm Formation and Swimming Motility.

To screen, identify and study the genes involved in isothiazolone resistance and biofilm formation in Citrobacter werkmanii strain BF-6. A Tn5 transposon library of approximately 900 mutants of C. werkmanii strain BF-6 was generated and screened to isolate 1,2-benzisothiazolin-3-one (BIT) resistant strains. In addition, the tRNA 2-thiocytidine (32) synthetase gene (ttcA) was deleted through homologous recombination and the resulting phenotypic changes of the ΔttcA mutant were studied. A total of 3 genes were successfully identified, among which ΔttcA mutant exhibited a reduction in growth rate and swimming motility. On the other hand, an increase in biofilms formation in ΔttcA were observed but not with a significant resistance enhancement to BIT. This work, for the first time, highlights the role of ttcA gene of C. werkmanii strain BF-6 in BIT resistance and biofilm formation.