Genome methylation library, preparation method and application thereof
A methylation and genome technology, applied in the field of genome methylation library and its preparation, can solve the problems of base imbalance, reduce the comparison rate of reads containing non-methylated cytosine, and cannot completely cover the CpG region, etc. Achieve the effect of improving the quality of sequencing and improving the base balance of the library
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[0034] The method for preparing a genome methylation library according to an embodiment of the present invention includes the following steps S1-S5:
[0035] S1, providing a transposase complex (transposome), the transposase complex is composed of a transposable adapter and a transposase, and the transposable adapter contains a transposon sequence;
[0036] S2. Using a transposase complex to fragment genomic DNA (tagmentation) to obtain a fragmented product with a transposable adapter and a gap;
[0037] S3, using dNTPs that replace dCTP with mdCTP to perform gap filling (gap filling) on the fragmented product;
[0038] S4, converting the fragmented product after the gap filling, so that the unmethylated cytosine is converted into uracil;
[0039] S5. Perform PCR amplification (amplification) on the transformed fragmented product to obtain a genome methylation library.
[0040] The present invention fragments the genomic DNA through the transposase complex and adds adapter...
Embodiment 1
[0058] Example 1 Genome methylation library construction mediated by Tn5 transposase
[0059] (1) Sample preparation: HEK293 cells were taken, and DNA was extracted using a cell genome DNA extraction kit. The resulting nucleic acids were dissolved in 50 μL TE buffer, and the concentration was measured using a Qubit 3.0 fluorometer. Finally, 50 ng DNA was taken for library construction according to the following operation steps.
[0060] (2) Tn5 processing mark:
[0061] a. Synthesize a nucleic acid fragment containing a 19bp transposase recognition sequence (the detailed sequence is shown in the following SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), wherein part of the cytosine in sequence B and sequence C is formazan Modified cytosine ( C ). In sequence B and sequence C, "N" represents any base A, T, C or G, wherein cytosine is methylated modified cytosine.
[0062] Linker sequence A: CTGTCTCTTATACACATCT (SEQ ID NO: 1);
[0063] Linker sequence B: T C GT C GG C AG ...
Embodiment 2
[0084] Example 2 Construction of genome methylation library mediated by Tn5 transposase and nick-digested
[0085] (1) Sample preparation: HEK293 cells were taken, and DNA was extracted using a cell genome DNA extraction kit. The resulting nucleic acids were dissolved in 50 μL TE buffer, and the concentration was measured using a Qubit 3.0 fluorometer. Finally, 50 ng DNA was taken for library construction according to the following operation steps.
[0086] (2) Tn5 processing mark:
[0087]a. Synthesize a nucleic acid fragment containing a 19bp transposase recognition sequence (the detailed sequence is shown in the following SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), wherein part of the cytosine in sequence B and sequence C is formazan Modified cytosine ( C ). In sequence B and sequence C, "N" represents any base A, T, C or G, wherein cytosine is methylated modified cytosine.
[0088] Linker sequence A: CTGTCTCTTATACACATCT (SEQ ID NO: 1);
[0089] Linker sequence B: T...
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