The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® has been tested in many sequencing applications including whole genome sequencing, hybrid capture, amplicon sequencing, and copy number assessment, etc. It has been designed to maximize the number of molecules incorporated into a sequencing library. As a result, this method is able to produce diverse libraries even from very small amounts of input DNA (< 1 ng). Please keep in mind the following:
- Modifications present in the DNA input, such as methylation and hydroxymethylation, can be replaced by their canonical form when using FS. This method should not be used for applications where modified bases are being studied (e.g. EM-seq and bisulfite sequencing).
- At very low frequency (typically less than 0.1%), FS may introduce artifacts identified as chimeras or tandem reads in downstream analysis. These artifacts can be filtered using fgbio FindSwitchbackReads.
- For FFPE samples, we have observed increased noise in some FS libraries. However, due to the diversity of FFPE damage, it is not possible to predict which FFPE samples will exhibit higher noise.
We encourage users to carefully evaluate any library prep method to ensure it is appropriate for the specific sample type being studied. Please reach out to [email protected] if you have questions about your specific application.