Library Preparation with FFPE DNA Samples

Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples is a common practice. However, the methods used for fixation and storage significantly damage the nucleic acids from these samples. Additionally, FFPE DNA samples are often available in only very small amounts. As a result, it can be challenging to construct high quality libraries in sufficient quantity to achieve good sequence data at the desired depth of coverage. NEBNext® Ultra™ II libraries were made from low nanogram amounts of several FFPE DNA samples of varying age and quality and then sequenced. Bioanalyzer traces of the libraries and analysis of the sequencing data show that the quality of the libraries was high.

NEBNext Ultra II produces the highest yield libraries from a broad range of input amounts

Kidney Tumor 17 132 91.5 96.1 0.48 3.0
Lung Tumor 20 232 90.1 94.9 0.42 4.1
Liver Normal 20 691 92.6 94.7 0.33 8.6
Breast Tumor 30 514 91.9 95.1 0.37 4.5



Libraries were prepared from 17–30 ng of human DNA extracted from the FFPE tissue samples listed, amplified using 10 cycles of PCR and sequenced on the Illumina MiSeq®. This data illustrates that NEBNext Ultra II enables high quality sequence data, even with low input amounts of FFPE DNA.

A: Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4.
% Mapped: The percentage of reads mapped to Human GRCh37 reference.
% Mapped in Pairs: The percentage of reads whose mate pair was also aligned to the reference.
% Duplication: The percentage of mapped sequence that is marked as duplicate.
% Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.

B: Bioanalyzer® traces of each library show high quality libraries with minimal adaptor-dimer.

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