16s rdna amplicon pyrosequencing  (Pyrosequencing Inc)

Journal: Microbial Biotechnology

Article Title: Microbial Communities Powering Plant‐Microbial Fuel Cells: Diversity, Functions and Biotechnological Perspectives

doi: 10.1111/1751-7915.70310

Figure Lengend Snippet: PRISMA 2020 flow diagram detailing study selection for the systematic review of rhizosphere microbiomes in plant‐microbial fuel cells (PMFCs). Final inclusion: 21 studies (2008–2025) reporting bacterial/archaeal 16S rRNA data from PMFC anode rhizospheres (Table ).

Article Snippet: 16S rRNA gene amplicon 454 pyrosequencing (bacteria), 16S rRNA gene Sanger‐based clone libraries (archaea) , Lu et al. ( ) .

Techniques: Selection

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a Diagram illustrating the induced aberrations by designer nucleases and the CLEAR-time dPCR assay strategies. b Absolute copy number and linkage normalisation workflow. Data are shown as mean ± s.d. c Genome copy frequency summarisation of on-target aberrations 3 h, 3- and 14-days post-Cas9 editing with and without AAV transduction targeting the WAS locus. Data are shown as mean ± s.d. d Single cleavage restriction digestion of AAV donor template VCN and % integrated donor template measured in AAV-transduced cells 3- and 14-days post-editing. Data are shown as mean ± s.d. e End trimming was measured as absolute loss of 5’- and 3’- sequences flanking the Cas9 cleavage site 3 h, 3- and 14-days post-Cas9 editing in RNP-only edited cells. Data are shown as mean ± s.d. f Aneuploidy measured as the absolute change of p or q arm copy numbers. Data are shown as mean ± s.d. g Validation of indel frequency by comparing the relative indel frequency calculated by dPCR, T7EI assay, ICE and NGS measured in WAS edited HSPCs 3-days post-editing. Abs. and Rel. refer to absolute and relative indels (i.e., normalised or not normalised to a reference), respectively. Data for T7EI, ICE, and NGS represent n = 1, dPCR data shown as mean ± s.d. of n = 3 technical replicates. h Validation of donor-template integration by comparing digital PCR to flow cytometry of AAV-transduced cells at 3- and 14-days post-editing. Flow cytometry data represent n = 1, dPCR data shown as mean ± s.d. of n = 3 technical replicates. i Qualitative validation of large deletion and other aberrations of RNP only edited cells 3-days post-editing using CAST-seq. On top, WAS gene schematic, exons in bold. Light blue indicates aberrations on negative-strand, light red indicates aberrations on positive-strand ( n = 2 technical replicates). j NGS targeted sequencing spanning ~2500 bp of the cleavage site targeting WAS (white arrowhead) indicating small and large deletions ( > 250 bp). X-axis indicates nucleotide position; Y-axis indicates number of mapped reads. Scale bar indicates 250 bp. All data represents n = 3 technical replicates unless stated otherwise. b , c , e One-way ANOVA with Sidak’s multiple comparison test. d Two-way ANOVA with Tukey multiple comparisons test. f Two-way ANOVA with Sidak’s multiple comparison test. n.s.= no statistical significance, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Transduction, Biomarker Discovery, T7EI Assay, Digital PCR, Flow Cytometry, Sequencing, Comparison

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a Schematic of wildtype and large deletion cassettes used to establish the LoD of large deletions, and representation of clonal expansion of cells harbouring a large deletion (orange) mediated genotoxic aberration amongst wildtype cells (blue). Dark and light grey bar indicates CCR5 and reference sequences, respectively. Red bar indicates flag sequence used to fuse assay and reference sequences. b Correlation of observed against the expected percentage of large deletions. Vertical black dotted line represents the limit of detection. Solid and dotted red line represents line of regression with 95% confidence interval, respectively. R 2 = 0.997. Calculated with linear regression analysis in GraphPad. Data shown as mean ± s.d. of n = 4 technical replicates. c CLEAR-time dPCR summaries of Cas9-edited HSPCs targeting various genes at different timepoints. The BTK edited HSPCs were also transduced with AAV6 encoding GFP. All editing was normalised against unedited mock electroporated HSPCs. Data are shown as mean ± s.d. d CLEAR-time dPCR summary of SH2D1A edited T cells with Cas9, Cas12, and TALENs at 3 days post-editing. Data are shown as mean ± s.d. e CLEAR-time dPCR summary of on-target CCR5 edited with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. f DSB and indels quantification at three known off-targets targeting CCR5 with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. g CLEAR-time dPCR on XIAP edited HSPCs pre-transplant and 16 weeks post-transplant. Data are shown as mean ± s.d. n = 4 mice for each treatment group. h Integration frequency in 16-week post-transplant XIAP edited and AAV-transduced hCD45 cells by dPCR and flow cytometry. Flow cytometry data represent n = 1 per mouse, dPCR data shown as mean ± s.d. of n = 3 technical replicates. i CLEAR-time dPCR normalised ICE analysis of pre-transplant XIAP edited HSPCs and post-transplant XIAP edited hCD45 cells. All data represents n = 3 technical replicates unless stated otherwise. d – f Two-way ANOVA with Tukey post-hoc test. n.s.= no statistical significance, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Sequencing, Transduction, TALENs, Flow Cytometry

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: Characterisation of aberrations induced by repair inhibitors on Cas9 edited HSPCs targeting the CCR5 locus. a Summary comparison between CLEAR-time dPCR (top) and ICE (bottom) 1, 3, and 14-days post-editing. Rel. indels = relative indels. For CLEAR-time dPCR, data are shown as mean ± s.d. For ICE, data are shown as n = 1. b Quantification of sequence trimming around the cleavage site in CCR5 edited HSPCs 1, 3, and 14-days post-editing. Data are shown as mean ± s.d. c CLEAR-time dPCR summary of aberrations induced by repair inhibitors on Cas9 edited iPSCs (top) and HSPCs (bottom) targeting the CD34 locus with and without an ssODN 3, 7, and 14-days post-editing. Data are shown as mean ± s.d. d Quantification of sequence trimming around the cleavage site of CD34 edited iPSCs (top) and HSPCs (bottom) 1, 3, and 14-days post-editing. Data are shown as mean ± s.d. e Comparison between ICE (I) and CLEAR-time dPCR (Ct) in Cas9 edited T cells (top), HSPCs (middle), and iPSCs (bottom) targeting the CCR5 locus with and without AAV transduction and AZD7648 treatment 4-days post-editing. Data are shown as mean ± s.d. ( n = 3 technical replicates of 2 independent donors per cell type). All data represents n = 3 technical replicates unless stated otherwise. b , d Two-way ANOVA with Tukey post-hoc test., n.s.= non-significant, **** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Comparison, Sequencing, Transduction

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a Overview of factors affecting nuclease-based editing activity. b Stacked bar-charts of observed dPCR timeseries genome copy frequency summaries of CD34 edited HSPCs using PsCas9 with and without repair inhibitors 5 minutes to 14 days post editing. Data are shown as mean ± s.d. c Updated three-state model of nuclease-induced cleavage and subsequent precise or mutation repair. Where; k dsb , k pr , k in , k ld , k ti =rate coefficients of DSBs, precise repair, indels, large deletions, and targeted integration, per h, respectively. D(t) = Cas9 nuclear trafficking delay based on the time in hours. d ODE fitted curves (lines) modelled using the first 24 h of CLEAR-time dPCR data. Dots and error bars indicate mean ± s.d. of observed dPCR data. e Kinetic activity rates per minute derived from the ODE fitted curves. V max =time at which maximum event velocity occurred. f Comparison of event V max in untreated and DSB repair-inhibited cells. g Comparison between untreated and repair-inhibited HSPCs precisely repaired genome copy frequency (top) and rate of precise repair (bottom). h Accumulation of DSBs and precisely repaired DNA in RNP-only edited cells in a 24-h time-period. Values in brackets indicates time at which DSBs exceeded 100% genome copies. i Model of recurring cleavage activity of precisely repaired DSBs. Blue and purple lines indicate frequency of DSBs and precise repairs, respectively. Black line indicates uncleaved sequences. j Comparison of rate coefficients, in events/h, estimated by the ODE kinetics between untreated and repair-inhibited cells. k Comparison of average half-life of DSBs to generate from uncleaved sequences and resolve as a repair product in untreated and repair inhibited HSPCs. l Pie-charts of the likelihood of DSB resolution into each measured repair product or remaining unresolved per hour. Calculated using the ratio of rate coefficients and the sum of all repair outcome rate coefficients. m Average number of DSBs necessary to generate each repair product. Calculated using the ratio of the specific repair product rate coefficient and the sum of the rate coefficient of all repair products. All data represents n = 3 technical replicates unless stated otherwise. All data derived from modelling ( n = 3 replicates, 1000 bootstraps/kinetic) is shown as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Activity Assay, Mutagenesis, Derivative Assay, Comparison

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a A stacked bar chart of observed dPCR timeseries genome copy frequency summaries of edited K562 cells using SpCas9 (blunt cut) and PsCas9 (staggered cut) with and without repair inhibitors/ssODN donor sequence, 5 min to 14 days post editing. Media change 24 h represented with vertical dotted bars. Data are shown as mean ± s.d. b ODE fitted curves (lines) modelled using the first 24 h of CLEAR-time dPCR data on SpCas9 (left) and PsCas9 (right) edited cells. Dots and error bars indicate mean ± s.d. of observed dPCR data. c Comparison of the average half-life of DSBs generated from uncleaved sequences and resolved as a repair product in untreated and repair-inhibited HSPCs. d Pie-charts of the likelihood of DSB resolution into each measured repair product or remaining unresolved per hour. e Average number of DSBs necessary to generate each repair product. Data are shown as mean ± s.d. f Stacked bar-charts of observed dPCR timeseries genome copy frequency summaries of edited T cells and K562 cells using SpCas9 with and without repair inhibitors/AAV-transduction 5 minutes to 14 days post editing. Data are shown as mean ± s.d. g ODE fitted curves (lines) modelled using the first 24 h of CLEAR-time dPCR data on T cells (left) and K562 cells (right) edited cells. Dots and error bars indicate mean ± s.d. of observed dPCR data. h Comparison of the average half-life of DSBs generated from uncleaved sequences and resolved as a repair product across all treatments in T cells (left) and K562 cells (right). i Pie-charts of the likelihood of DSB resolution into each measured repair product or remaining unresolved per hour. Calculated using the ratio of rate coefficients and the sum of all repair outcome rate coefficients. j Average number of DSBs necessary to generate each repair product. All data represents n = 3 technical replicates unless stated otherwise. All data derived from modelling ( n = 3 replicates, 1000 bootstraps per condition) is shown as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Sequencing, Comparison, Generated, Transduction, Derivative Assay

Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: Graphical representation of designer nuclease cleavage and cellular repair. Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript). CLEAR-time dPCR data are shown as mean ± upper/lower limits of 95% CI ( n = 3 replicates, 1000 bootstraps per condition) linked to Figs. and . Data points from published literature shown as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Bar charts illustrate the rate coefficients calculated through the different phases of the DNA cleavage and repair process in comparisons with previously published articles addressing the DNA repair kinetics with Sanger sequencing + amplicon deep sequencing + LM-PCR (Brinkman et al.), LM-PCR (Ben-Tov et al.), and dPCR + amplicon deep sequencing (Liu et al.) vs. CLEAR-time dPCR (this manuscript).

Techniques: Sequencing, Amplification