Hello!
I am trying to optimise a protocol for low cell number input for RRBS library prep. I am having an ongoing issue where the library prep appears to be successful, when checking the FASTQC results there are high quality read scores, good traces indicating MSPI digestion and bisulfite conversion worked and overall the signatures look very similar to our previous bulk (larger input) RRBS libraries which have been successful. However when the libraries are aligned we are consistently only getting 1-5% alignment (even in our larger input libraries 500-1000 cells), these are human cells by the way. We have not identified any contaminant through a blast search and I have now individually tested and combined each of the processes (e.g lysis, MSPI digestion, adapter ligation, and bisulfite conversion) which all appear to be working effectively. I've altered the adapter dilution, and trialled different polymerases, all these conditions appear to have no effect on improving the final output. Has anyone had any similar issues? Or any advice as to what to tackle next?
I have attached the protocol paper which I am following for reference.
I am trying to optimise a protocol for low cell number input for RRBS library prep. I am having an ongoing issue where the library prep appears to be successful, when checking the FASTQC results there are high quality read scores, good traces indicating MSPI digestion and bisulfite conversion worked and overall the signatures look very similar to our previous bulk (larger input) RRBS libraries which have been successful. However when the libraries are aligned we are consistently only getting 1-5% alignment (even in our larger input libraries 500-1000 cells), these are human cells by the way. We have not identified any contaminant through a blast search and I have now individually tested and combined each of the processes (e.g lysis, MSPI digestion, adapter ligation, and bisulfite conversion) which all appear to be working effectively. I've altered the adapter dilution, and trialled different polymerases, all these conditions appear to have no effect on improving the final output. Has anyone had any similar issues? Or any advice as to what to tackle next?
I have attached the protocol paper which I am following for reference.
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