Journal: Journal of Clinical Immunology

Article Title: Immunologic and Genetic Contributors to CD46-Dependent Immune Dysregulation

doi: 10.1007/s10875-023-01547-y

Figure Lengend Snippet: Exon 4 partial skipping and transcript coexistence of mutated CD46 . a Representative Sanger sequencing of a PCR-amplified CD46 -specific sequence using PBMC-derived genomic DNA. Purple arrow shows the heterozygous CD46 intronic mutation detected in five out of six tested family members. b PCR amplification of a CD46 -specific sequence using PBMC-derived cDNA revealing two different bands in all tested individuals except family member number 3 which does not carry the CD46 mutation in genomic DNA. Image shown is zoomed for bands and ladder. c Sanger sequencing chromatogram of the bands indicated in b show a skipping of 21 nucleotides in the truncated cDNA band from the del21bp CD46 gene (bottom). d mRNA expression quantitation of WT (top) and del21bp mutant (bottom) CD46 mRNA assessed using PBMC-derived cDNA. Each are normalized as listed on the Y axis. P values represent comparison of all five CD46 mutation carriers and three healthy controls (HCs) by unpaired t tests

Article Snippet: The NIHR Bioresource Rare Disease project has provided whole genome sequencing data for more than 10’000 individuals with rare diseases [ ].

Techniques: Sequencing, Amplification, Derivative Assay, Mutagenesis, Expressing, Quantitation Assay, Comparison

Journal: Plant Methods

Article Title: A novel hairpin library-based approach to identify NBS–LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

doi: 10.1186/s13007-017-0181-7

Figure Lengend Snippet: Schematics of the procedure to identify NBS domain-containing proteins in N. benthamiana annotated genome. HMMsearch of N. benthamiana annotated proteins using the standard Pfam NBS HMM led to the identification of 309 NBS-containing candidates. The 309 NBS domains were aligned using ClustalW and used to build a N. benthamiana -specific NBS HMM, which was subsequently used to screen again the N. benthamiana annotated proteins. However, no new candidates were identified in the 268 entries generated. N. benthamiana annotated proteins were further searched by BLASTp using the 309 candidates. New R gene candidates were further selected and screened for the presence of NBS, LRR and TIR domains using HMMscan

Article Snippet: Taking advantage of the availability of the N. benthamiana genome (Boyce Thompson Institute for Plant Research—Genbank: PRJNA170566), sequences of most of the potential R genes of the N. benthamiana genome were identified based on the presence of an NBS domain and used to create an RNAi knock-out library.

Techniques: Generated

Journal: Plant Methods

Article Title: A novel hairpin library-based approach to identify NBS–LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

doi: 10.1186/s13007-017-0181-7

Figure Lengend Snippet: Hairpin library design. a A total of 281 Kmers were designed, with 202 Kmers targeting a unique gene of N. benthamiana annotated genome with 100% identity, and 79 kmers targeting two or more genes. b To produce multiple gene silencing constructs, groups of six kmers were synthesized in tandem (4 “singles” and 2 “multiple”), cloned in a Gateway-enabled pUC57 vector and eventually recombined by LR reaction into the hairpin-producing destination vector pTKO2

Article Snippet: Taking advantage of the availability of the N. benthamiana genome (Boyce Thompson Institute for Plant Research—Genbank: PRJNA170566), sequences of most of the potential R genes of the N. benthamiana genome were identified based on the presence of an NBS domain and used to create an RNAi knock-out library.

Techniques: Construct, Synthesized, Clone Assay, Plasmid Preparation

Journal: Plant Methods

Article Title: A novel hairpin library-based approach to identify NBS–LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

doi: 10.1186/s13007-017-0181-7

Figure Lengend Snippet: Pto/avrPto-triggered HR is cancelled by hp#12 pre-infiltration. a General design of the assay: N. benthamiana leaf was infiltrated with Agrobacterium containing the hairpin construct or corresponding empty vector (EV) in two distinct patches on the right-hand side and left hand side of the leaf respectively and infiltration areas were marked with a felt-tip pen. The following day, Agrobacterium containing the effector construct, or corresponding empty vector (EV), were infiltrated in the pre-infiltrated patches of the top half and bottom half of the leaf respectively. Development of the HR was monitored during the following 3–7 days depending on the strength of the HR. b Agrobacterium suspension (OD 0.2) carrying the hairpin hp#12 or corresponding empty vector was infiltrated in N. benthamiana leaf and the Agrobacterium suspension (OD 0.2) expressing the Pto/avrPto construct was infiltrated 24 h later on the pre-infiltrated patches as designed in ( a ). Photos was taken 4 days after Pto/avrPto infiltration

Article Snippet: Taking advantage of the availability of the N. benthamiana genome (Boyce Thompson Institute for Plant Research—Genbank: PRJNA170566), sequences of most of the potential R genes of the N. benthamiana genome were identified based on the presence of an NBS domain and used to create an RNAi knock-out library.

Techniques: Construct, Plasmid Preparation, Suspension, Expressing

Journal: Plant Methods

Article Title: A novel hairpin library-based approach to identify NBS–LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

doi: 10.1186/s13007-017-0181-7

Figure Lengend Snippet: TMV-induced hypersensitive response (HR) in N -infiltrated leaves is cancelled by NRG1 silencing. Hairpins hp#26 or u135 or the corresponding empty vector were co-infiltrated with N in N. benthamiana leaves and the infiltrated areas were marked with a felt-tip pen. TMV was inoculated 24 h after Agrobacterium infiltration. HR was visible from 4 days after TMV inoculation and photo were taken 6 days post-inoculation. H 2 O 2 production in the same leaves was monitored using DAB staining

Article Snippet: Taking advantage of the availability of the N. benthamiana genome (Boyce Thompson Institute for Plant Research—Genbank: PRJNA170566), sequences of most of the potential R genes of the N. benthamiana genome were identified based on the presence of an NBS domain and used to create an RNAi knock-out library.

Techniques: Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: Accurate cross-species 5mC detection for Oxford Nanopore sequencing in plants with DeepPlant

doi: 10.1038/s41467-025-58576-x

Figure Lengend Snippet: CHH methylation sites, particularly those with high-methylation levels (≥90%), are rare in plants. This figure presents the statistics on high-methylation CHH sites from previously published bisulfite sequencing (BS-seq) datasets , – and those generated in this study. a Ratios of high-methylation CHH sites among quantified CHH motifs (≥5 read coverage) (left panel) and the number of covered 9-mer contexts (right panel) in BS-seq datasets from ten plant species. Only 9-mers observed at three or more high-methylation CHH sites were considered. Species other than A. thaliana and O. sativa were selected based on high CHH methylation ratios reported in previous studies , . b Ratios of high-methylation CHH sites in BS-seq datasets sequenced for this study, including A. thaliana , O. sativa , and species with abundant high-methylation CHH sites identified in ( a ), as well as Glycine max and Marchantia polymorpha . c Number of covered 9-mer contexts (top) and heatmap of context abundance (bottom) grouped by CHH motifs in nanopore datasets from six plant species. In the top panel, the top line of each bar corresponds to the number (36,864) of all possible 9-mer sequences centered with a CHH motif. A 9-mer was considered covered if present in 50 or more positive training samples and had at least an equal number of negative samples. “Mixed” refers to combined samples from S. miltiorrhiza , R. communis , and S. tuberosum . Source data are provided as a Source Data file.

Article Snippet: After bisulfite conversion, PCR amplification was performed, and the sequencing data were generated by BGI Genomics Co. Ltd.

Techniques: Methylation, Methylation Sequencing, Generated

Journal: Nature Communications

Article Title: Accurate cross-species 5mC detection for Oxford Nanopore sequencing in plants with DeepPlant

doi: 10.1038/s41467-025-58576-x

Figure Lengend Snippet: Quantitative evaluation of CHH methylation detection by DeepPlant and Dorado

Article Snippet: After bisulfite conversion, PCR amplification was performed, and the sequencing data were generated by BGI Genomics Co. Ltd.

Techniques: Methylation, Sequencing

Journal: Nature Communications

Article Title: Accurate cross-species 5mC detection for Oxford Nanopore sequencing in plants with DeepPlant

doi: 10.1038/s41467-025-58576-x

Figure Lengend Snippet: a Circos plot illustrating DeepPlant CHH methylation profiling in centromeric regions and 100 kb intervals upstream and downstream in O. sativa . From outer to inner: ideograms of centromere (center box) and neighboring regions (two terminal boxes); histograms of normalized sequencing coverage across 100 bp bins (gray, normalized against mean genomic coverage); histograms of CHH methylation frequencies (blue) across 100 bp bins. b Comparison of CHH motif coverage ratios across different genomic regions between BS-seq, DeepPlant, and Dorado profiling. To be noticed, the same nanopore dataset was used for DeepPlant and Dorado profiling, and the coverage difference between DeepPlant and Dorado derived from the distinct read filters they applied. DeepPlant applies three thresholds for screening high-quality alignments, including MAPQ ≥ 20, primary alignment length/read length ≥80%, and mapping identity ≥80% by default. Only CHH motifs with a minimum read coverage of 10 were regarded as quantified. TE transposable element, CDS non-TE protein-coding sequences. c Read coverage and CHH methylation frequencies in the centromeric regions of Chr1, comparing whole-genome BS-seq data, DeepPlant, and Dorado analysis on Nanopore data. d Boxplot illustrating CHH methylation frequencies on the forward (+) and reverse (−) strands in protein-coding and transposable element (TE) regions. The center line represents the median; each box shows the first and third quartiles; minima represents the larger between Q1−1.5×IQR and the minimum observed value; maxima represents the smaller between Q3 + 1.5×IQR and the maximum observed value. e Violin plot displaying strand-specific CHH methylation status across various TE types and non-TE protein-coding regions. The annotation of TEs and protein-coding regions was acquired from T2T-NIP . Source data of ( c , d ) are provided in Zenodo [10.5281/zenodo.15062213]. Source data of the other panels are provided as a Source Data file.

Article Snippet: After bisulfite conversion, PCR amplification was performed, and the sequencing data were generated by BGI Genomics Co. Ltd.

Techniques: Methylation, Sequencing, Comparison, Derivative Assay