Home FAQs Can libraries be prepared from FFPE DNA using the NEBNext® EM-seq Kit (NEB #E7120)?

FAQ: Can libraries be prepared from FFPE DNA using the NEBNext® EM-seq Kit (NEB #E7120)?

Yes, libraries can be prepared using 10-200 ng FFPE DNA inputs. The DNA should be sheared and the libraries constructed according to the EM-seq Manual. PCR cycles may need to be optimized, but we recommend increasing by at least 2 cycles for the 10 ng and 50 ng inputs (i.e., for 10 ng use at least 10 PCR cycles and for 50 ng use at least 8 PCR cycles).

Note that libraries prepared from FFPE DNA may have higher methylation backgrounds in the CHH and CHG contexts. However, use of a 3xC filter (available in the Bismark pipeline) or similar, reduces background. The filter removes reads that contain 3 or more consecutive unconverted Cs in the non-CpG context (i.e., CHG/CHH).