We seem to be having some difficulties creating reproducible ddRADSeq libraries.
We are noticing that after running the first of the processing steps (process radtags) - which removes the barcodes, filters for the rad sites, and checks for sequence quality - we have some evidence of perhaps incomplete restriction digestion.
For instance - we have:
rad cut site (i.e, CATG from NlaIII) - followed by target sequence - followed by the second cut site (i.e., MluCI - AATT) - followed by more sequence.
Shouldn't the sequence end at the second cut site? Has anyone tried a sequential digest? We have been running the test libraries at 150bp SE on a MiSeq.
Any help is greatly appreciated.
We are noticing that after running the first of the processing steps (process radtags) - which removes the barcodes, filters for the rad sites, and checks for sequence quality - we have some evidence of perhaps incomplete restriction digestion.
For instance - we have:
rad cut site (i.e, CATG from NlaIII) - followed by target sequence - followed by the second cut site (i.e., MluCI - AATT) - followed by more sequence.
Shouldn't the sequence end at the second cut site? Has anyone tried a sequential digest? We have been running the test libraries at 150bp SE on a MiSeq.
Any help is greatly appreciated.
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