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Hi All,
almost all sequencing library preparation kit providers now offer kits promising "random" enzymatic DNA fragmentation that are not based on transposon tagmentation - adapters are ligated to the fragments afterwards.
How can this be achieved?
This is one of the aspects of the library prep chemistry that escapes me.
Early protocols might have used Benzonase (e.g. Shearase?). DNAse I in combination with a second nuclease completing the double-strand break might have been used for the first NEB Fragmentase. Another option might be the use of a "balanced" cocktail of restriction enzymes?
How can the reaction be controlled to arrive at usable fragment lengths and allow a mostly unbiased representative representation of the sample in the resulting library?
The degree of fragmentation is for some kits clearly DNA sample concentration dependent but some providers claim their reactions are merely time-dependent (e.g. Nugen , NEB) which would be very advantageous, if true.
almost all sequencing library preparation kit providers now offer kits promising "random" enzymatic DNA fragmentation that are not based on transposon tagmentation - adapters are ligated to the fragments afterwards.
How can this be achieved?
This is one of the aspects of the library prep chemistry that escapes me.
Early protocols might have used Benzonase (e.g. Shearase?). DNAse I in combination with a second nuclease completing the double-strand break might have been used for the first NEB Fragmentase. Another option might be the use of a "balanced" cocktail of restriction enzymes?
How can the reaction be controlled to arrive at usable fragment lengths and allow a mostly unbiased representative representation of the sample in the resulting library?
The degree of fragmentation is for some kits clearly DNA sample concentration dependent but some providers claim their reactions are merely time-dependent (e.g. Nugen , NEB) which would be very advantageous, if true.