FFPE DNA Input Recommendations

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The Illumina FFPE DNA Prep with Exome 2.5 Enrichment is optimized to prepare libraries from gDNA that is fragmented to 100–350 bp, and requires 40 ng of FFPE DNA input. Inputs lower than 40 ng can affect assay performance. Use a Nucleic acid isolation method that produces high recovery yields, minimizes sample consumption, and preserves sample integrity like the QIAGEN AllPrep DNA/RNA FFPE Kit. Quantify the input DNA before beginning the protocol. Use a fluorometric quantification method that uses DNA binding dyes, such as the Qubit Fluorometer 4.0 or Accuclear.

Libraries prepared with the Illumina FFPE DNA Prep with Exome 2.5 Enrichment are compatible with NovaSeq 6000 RUO, NovaSeq X, and NextSeq 2000 sequencing systems. For information on sequencing compatibility and run settings, refer to the Illumina FFPE DNA Prep with Exome 2.5 Enrichment support page on the Illumina support website.

Use reference materials with known variant compositions. For example, use Seraseq Tumor Mutation DNA mix v2 AF10 as tumor, and Seraseq TNA WT mix (DNA only) as matched normal.

Processing reference samples reduces the total number of test samples that can be processed.

For optimal performance, assess FFPE DNA sample quality by using the Illumina TruSight FFPE QC kit. Before using the Illumina FFPE DNA Prep with Exome 2.5 Enrichment, dilute the FFPE DNA to 1 ng/µl.

Use DNA samples that result in delta Cq value ≤ 4. Processing samples with delta Cq value > 4 might result in decreased coverage. Performance of poor quality DNA samples may be improved with deeper sequencing and/or increased input (50 ng).

The assay is tested using the LE220-plus/ME220/R230 Covaris Focused-ultrasonicator with the parameters provided in Fragment FFPE DNA. Fragment size distribution can vary due to differences in sample quality and the sonication instrument used for fragmentation. Use the following guidelines for shearing.

Avoid excessive bubbles or an air gap in the shearing tube as it can lead to incomplete shearing.

Load the DNA into the Covaris tube slowly to avoid creating bubbles.

Centrifuge the Covaris tube to collect the sample at the bottom of the tube before shearing.

If using the LE220-plus/ME220/R230 Covaris instrument, fill unused wells with 52 µl TE buffer (TEB) to provide substance for optimal machine performance.

[Optional] Assess fragment DNA size distribution of sheared samples using the Agilent DNA ScreenTape Analysis (D1000) with the Agilent 4200 TapeStation System.