Journal: Genome Biology

Article Title: NT-seq: a chemical-based sequencing method for genomic methylome profiling

doi: 10.1186/s13059-022-02689-9

Figure Lengend Snippet: Principle and workflow of NT-seq. a Schematic illustration of nitrite treatment. Nitrite treatment induces deamination of adenine, cytosine, and 5mC at different frequencies, producing inosine, uracil, and thymine, respectively. Meanwhile, nitrite treatment nitrosylates 6mA and 4mC, producing nitrosylated 6mA (6mA-NO) and nitrosylated 4mC (4mC-NO). During PCR amplification and sequencing, base pairing and reading for each product are labeled on the right column. b The workflow of NT-seq. Single-stranded DNA is first annealed with protective oligos to protect PCR primer regions. Annealed DNA is treated with nitrite and then amplified to construct the sequencing library. Sequencing data from native DNA and PCR control are used to calculate the A to G or C to T mutation ratio and to call methylation

Article Snippet: Illumina TruSeq adaptor was added to oligo by PCR amplification using Taq DNA polymerase (NEB, M0490S) (cycle number was determined by qPCR with iTaq polymerase (BioRad, 1725121)).

Techniques: Amplification, Sequencing, Labeling, Construct, Mutagenesis, Methylation

Journal: Genome Biology

Article Title: NT-seq: a chemical-based sequencing method for genomic methylome profiling

doi: 10.1186/s13059-022-02689-9

Figure Lengend Snippet: NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli MG1655 genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)

Article Snippet: Illumina TruSeq adaptor was added to oligo by PCR amplification using Taq DNA polymerase (NEB, M0490S) (cycle number was determined by qPCR with iTaq polymerase (BioRad, 1725121)).

Techniques: Methylation, Amplification, Sequencing, MANN-WHITNEY