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: medRxiv

Article Title: Optimizing culture-free approaches to recover high quality M. tuberculosis genomic variation

doi: 10.64898/2025.12.16.25342406

Figure Lengend Snippet: a) We conducted a serial dilution experiment in which we mixed two Mtb DNA standards representing lineage 4.1.2.1 (strain X003899) and 4.9 (strain H37ra), such that the minority strain represented 0-100% of the total DNA and then diluted Mtb DNA in human DNA, to simulate clinical samples which are overwhelmingly human DNA. We approximated the proportion of Mtb DNA found in sputum with smear microscopy grades 1+, 2+, and 3+. 7 We used hybrid capture to enrich for Mtb DNA, conducted whole genome sequencing of captured libraries on an Illumina platform. b) We additionally prospectively sampled matched Mtb diagnostic cultures and unprocessed sputum submitted to ARUP diagnostic laboratories. We performed hybrid capture and Illumina whole genome sequencing on matched samples.

Article Snippet: We then diluted the Mtb DNA with a human DNA standard (HTB-22D, ATCC HTB-22), so that total Mtb DNA comprised a small proportion of total input DNA by molecular weight (0.01%, 0.03%, 0.3%), approximating the proportion of Mtb DNA found in sputum with smear microscopy grades 1+, 2+, and 3+.

Techniques: Serial Dilution, Microscopy, Sequencing, Diagnostic Assay

Journal: medRxiv

Article Title: Optimizing culture-free approaches to recover high quality M. tuberculosis genomic variation

doi: 10.64898/2025.12.16.25342406

Figure Lengend Snippet: (a) The percentage of reads taxonomically classified to the Mycobacterium genus by Kraken, (b) the percentage of reads mapping to Mtb after filtering, and mean coverage. The x-axis for both (a, b, and c) indicates the percentage input Mtb DNA in the total DNA mixture and color indicates the proportion H37Ra (color). (d) Precision and recall (sensitivity) of SNP identification, by percentage of M. tuberculosis , for the samples comprised of 100% Lineage 4.1.2.1 (points) and 100% 4.9 (triangles). Colors indicate genomic region: outside PE/PPE genes, blue; within PE/PPE genes, red.

Article Snippet: We then diluted the Mtb DNA with a human DNA standard (HTB-22D, ATCC HTB-22), so that total Mtb DNA comprised a small proportion of total input DNA by molecular weight (0.01%, 0.03%, 0.3%), approximating the proportion of Mtb DNA found in sputum with smear microscopy grades 1+, 2+, and 3+.

Techniques:

Journal: medRxiv

Article Title: Optimizing culture-free approaches to recover high quality M. tuberculosis genomic variation

doi: 10.64898/2025.12.16.25342406

Figure Lengend Snippet: Proportion minor strain (expected minor allele frequency) versus observed minor allele frequency for minority variants identified, with columns indicating the percentage of Mtb DNA in the input DNA before hybrid capture and rows indicating genomic location. Colors indicate minor allele status.

Article Snippet: We then diluted the Mtb DNA with a human DNA standard (HTB-22D, ATCC HTB-22), so that total Mtb DNA comprised a small proportion of total input DNA by molecular weight (0.01%, 0.03%, 0.3%), approximating the proportion of Mtb DNA found in sputum with smear microscopy grades 1+, 2+, and 3+.

Techniques: