Journal: iScience

Article Title: A targeted DNA methylation method for detecting gastrointestinal cancer in circulating cell-free DNA

doi: 10.1016/j.isci.2025.114342

Figure Lengend Snippet: Schematic and performance of the tMCTA-seq method (A) The tMCTA-seq begins with bisulfite treatment, converting unmethylated CpG tandems in CGIs to UpG tandems, leaving methylated CGIs unaffected. In the first step, primer A, which comprises a semi-random sequence with a CpG site at the 3′-end, a unique molecular identifier (UMI) sequence in the middle, and an anchor sequence at the 5′-end, anneals semi-randomly to converted DNA and extends. This step preferentially amplifies methylated CGIs due to their higher density of methylated CpG sites. Next, a set of locus-specific primers is used for targeted amplification in combination with primer TA (sharing partial sequence homology with primer A). Primer B, containing the “CGCGCGG” sequence, serves as a universal inner nested primer to selectively amplify methylated CpG tandem sites. Finally, indexed primers C and D were employed for PCR amplification to generate the sequencing library. (B) The input and captured molecules of 110 mCGCGCGG-CpG (upper: enlarged view of the dashed areas). The FMG was diluted from 150 pg (equivalent to 110 × 150 ÷ 3.3 = 5,000 molecules for the total 110 sites) down to 2.3 pg (equivalent to 110 × 2.3 ÷ 3.3 = 77 molecules for the total 110 sites) and spiked into 6 ng of WBC gDNA. (C) The captured molecules, and (D) sequencing depth of 110 mCGCGCGG-CpGs in MCTA-seq and tMCTA-seq, with 150 pg of FMGs spiked into 6 ng of WBC gDNA. Data are represented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, two-tailed Mann–Whitney-Wilcoxon test.

Article Snippet: Fully methylated human genomic DNA (FMG) , Zymo Research , D5014.

Techniques: Methylation, Sequencing, Amplification, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A targeted DNA methylation method for detecting gastrointestinal cancer in circulating cell-free DNA

doi: 10.1016/j.isci.2025.114342

Figure Lengend Snippet: Schematic and performance of the tMCTA-seq method (A) The tMCTA-seq begins with bisulfite treatment, converting unmethylated CpG tandems in CGIs to UpG tandems, leaving methylated CGIs unaffected. In the first step, primer A, which comprises a semi-random sequence with a CpG site at the 3′-end, a unique molecular identifier (UMI) sequence in the middle, and an anchor sequence at the 5′-end, anneals semi-randomly to converted DNA and extends. This step preferentially amplifies methylated CGIs due to their higher density of methylated CpG sites. Next, a set of locus-specific primers is used for targeted amplification in combination with primer TA (sharing partial sequence homology with primer A). Primer B, containing the “CGCGCGG” sequence, serves as a universal inner nested primer to selectively amplify methylated CpG tandem sites. Finally, indexed primers C and D were employed for PCR amplification to generate the sequencing library. (B) The input and captured molecules of 110 mCGCGCGG-CpG (upper: enlarged view of the dashed areas). The FMG was diluted from 150 pg (equivalent to 110 × 150 ÷ 3.3 = 5,000 molecules for the total 110 sites) down to 2.3 pg (equivalent to 110 × 2.3 ÷ 3.3 = 77 molecules for the total 110 sites) and spiked into 6 ng of WBC gDNA. (C) The captured molecules, and (D) sequencing depth of 110 mCGCGCGG-CpGs in MCTA-seq and tMCTA-seq, with 150 pg of FMGs spiked into 6 ng of WBC gDNA. Data are represented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, two-tailed Mann–Whitney-Wilcoxon test.

Article Snippet: The fully methylated human genomic DNA (FMG) was purchased from Zymo Research (D5014).

Techniques: Methylation, Sequencing, Amplification, Two Tailed Test, MANN-WHITNEY

Journal: Nature methods

Article Title: Coupling CRISPR Scanning with Targeted Chromatin Accessibility Profiling using a Double-Stranded DNA Deaminase

doi: 10.1038/s41592-025-02811-2

Figure Lengend Snippet: a) Sequence logo showing the sequence context preference for DddA11 on naked DNA. b) Aggregate profile of cytidine deamination fractions ( y axis) around DNase I hypersensitive sites (DHSs) from whole-genome sequencing (WGS) of DddA11-treated K562 cells. c) Bar plots comparing modified cytosine profiles between DddA11-WGS and NOMe-seq. 23 Left: Number of modified cytosines in open chromatin. Middle: Total number of modified cytosines detected genome-wide. Right: Fraction of modified cytosines overlapping CREs. d) Bar plot showing C•G-to-T•A editing rates at unmethylated, methylated, or untreated cytidines in CpG sites of varying sequence contexts in the chr7:27158522–27163197 region. e) Histogram showing the number of C•G-to-T•A mutations per read at the HS2 locus (chr11:5279265–5282582) in DddA11-free and DddA11-treated samples. The dashed line represents a 2% threshold of all candidate cytosines. f) Histogram of edit differences between randomly sampled read pairs at the HS2 locus in DddA11-free and DddA11-treated samples. The dashed line represents a 2% threshold of all candidate cytosines. g) Genome tracks displaying ATAC-seq, TDAC-seq, and DADs across indicated genomic regions. TDAC-seq signal represents the average number of DddA11 edits in a 50-bp window. h) TDAC-seq at the HS2 locus under varying DddA11 concentrations and reaction times, compared with ATAC-seq. TDAC-seq signal represents the average number of DddA11 edits in a 50-bp window. i) Schematic of hidden Markov model used to call DADs. j) Scatter plot showing the percentage of bases covered by both DAD signal and ATAC-seq peaks ( x -axis) versus bases covered only by DAD signal ( y -axis). The dashed diagonal line represents equal coverage. Each point represents TDAC-seq from a different locus. k) Top: genome tracks showing chromatin profiles, TDAC-seq, and DADs at the ZBTB38 locus. Bottom: heatmap showing DAD co-accessibility, shown as observed minus expected, calculated as the product of individual accessibility fractions. Data in b,g,j, and k are representative examples of n = 2 replicates. Correlation between TDAC-seq replicates is presented in Supplementary Fig. 1 .

Article Snippet: A total of 1 μg of pooled barcoded libraries was prepared, and final nanopore libraries were completed following the Ligation Sequencing V14 protocol (SQK-LSK114) with end-prep and adapter ligation, followed by sequencing on a PromethION Flow Cell (R10.4.1) with super accuracy (SUP, v4.3.0) base calling model. A 12.5 μL DddA11 reaction was prepared as described above, using 500 ng of either methylated or unmethylated human genomic DNA (Zymo Research, D5014).

Techniques: Sequencing, Modification, Genome Wide, Methylation

Journal: Nature methods

Article Title: Coupling CRISPR Scanning with Targeted Chromatin Accessibility Profiling using a Double-Stranded DNA Deaminase

doi: 10.1038/s41592-025-02811-2

Figure Lengend Snippet: a) Line plot showing the fraction of reads ( y axis) containing a deletion at the indicated position across the HS2 region ( x axis), with expected cut sites for each sgRNA indicated with red dotted lines. b) The number of deduplicated reads ( y axis) per genotypes ( x axis) from HS2 CRISPR deletions. In the HS2 Cas9 TDAC-seq sgRNA tiling library, 541 genotypes have at least 400 reads (red line). c) 2D histogram of UMIs plotted by the number of reads of the most abundant CRISPR genotype per UMI ( x axis) and all genotypes ( y axis). Both values would be equal for pure UMIs, indicated by red boxes along the diagonal. Data are representative of a sample of 10,000 sequencing reads. d) Heatmaps of TDAC-seq using CRISPR-Cas9 cutting on the HS2 enhancer in K562 cells. Individual sequencing reads were assigned to one of 541 genotypes ( y axis), with black lines indicating the CRISPR deletion position and length on the HS2 region. For each genotype, the average number of DddA11 edits in a 100-bp sliding window is shown across the HS2 region ( x axis). e) Heatmap showing the statistical significance (Welch’s two-sided t -test) of comparing DddA accessibility between reads that contain a deletion spanning the entire deletion window and all other reads. This value is calculated for all possible deletion windows indicated by their start position (horizontal axis) and length (vertical axis). Those with less than 100× coverage were excluded. Arrow indicates a 10-bp deletion window corresponding to the minimal key motif discussed in Fig. 4 . f) The proportion of reads from gDNA with and without CRISPR deletions after individual transduction of sgRNA-1 and sgRNA-11 into K562 cells. Data in a - e are representative examples of n = 2 replicates. Correlation between TDAC-seq replicates is presented in Supplementary Fig. 3 . Experiments for data in f were performed once.

Article Snippet: A total of 1 μg of pooled barcoded libraries was prepared, and final nanopore libraries were completed following the Ligation Sequencing V14 protocol (SQK-LSK114) with end-prep and adapter ligation, followed by sequencing on a PromethION Flow Cell (R10.4.1) with super accuracy (SUP, v4.3.0) base calling model. A 12.5 μL DddA11 reaction was prepared as described above, using 500 ng of either methylated or unmethylated human genomic DNA (Zymo Research, D5014).

Techniques: CRISPR, Sequencing, Transduction