While sufficient yield of a library is required for successful sequencing, quantity alone is not enough. The quality of a library is also critical, regardless of the input amount or GC content of the sample DNA. A high quality library will have uniform representation of the original sample, as well as even coverage across the GC spectrum.
Uniform GC Coverage
Libraries from varying input amounts of three microbial genomic DNAs with low, medium and high GC content (H. influenza, E. coli and H. palustris) were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit. In all cases, uniform coverage was obtained, regardless of GC content and input amount (A). GC coverage of libraries prepared using other commercially available kits was also analyzed using the same trio of genomic DNAs. Again, NEBNext Ultra II provided good GC coverage (B).