DNA Input Recommendations

The Illumina 5-Base DNA Prep with Enrichment protocol enables library preparation from gDNA or cfDNA input.

gDNA Input

The Illumina 5-Base DNA Prep with Enrichment protocol enables library preparation from 50–100 ng gDNA.

The input preparation procedure requires sonication of gDNA samples.

Follow these guidelines for sonication.

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Use a sonication method that produces an average fragment size of ~450 bp. Overfragmenting can reduce sample yield, introduce GC bias, and affect assay performance.

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Sonication reaction must be less than 100 µl to be compatible with this workflow.

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Optimize sonication for the sample type, sonication equipment, and sonication consumables.

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Illumina tested the Covaris LE220 Plus with the 8 microTUBE Strip for sonication with the following settings:

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Sonication volume: 55 µl

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Water temperature setpoint: 7°C, Range ± 2°C

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Peak incident power: 450 Watts

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Duty factor: 18%

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Cycles per burst: 200 cpb

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Treatment time: 35 seconds

For instructions on how to sonicate gDNA samples, refer to the appropriate supporting material for your sonication equipment.

cfDNA Input

The Illumina 5-Base DNA Prep with Enrichment protocol enables library preparation from 1–20 ng cfDNA.

Prepare Size-Select cfDNA includes bead purification. Bead purification is an essential preparation step. This step is optimized to select the mononucleosomal cfDNA peak and to remove blood-derived components that can interfere with library prep chemistry.

Follow these guidelines for input:

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Use one of the following tested blood collection tubes: the Apostle MiniMax Cell-Free DNA Blood Collection Tube (BCT), QIAGEN PAXgene Blood ccfDNA Tubes, Streck Cell-Free DNA BCT, or BD Vacutainer Plastic Blood Collection Tubes with K2EDTA.

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Use a nucleic acid isolation method that produces high recovery yields, minimizes sample consumption, and preserves sample integrity. The QIAGEN QIAamp Circulating Nucleic Acid Kit (without carrier RNA), QIAGEN QIAamp MinElute ccfDNA Midi Kit, and the Zymo MAGicBead cfDNA Isolation Kit are compatible with the Illumina 5-Base DNA Prep with Enrichment Kit.

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Quantify the input of each cfDNA sample using a size-based quantification method such as the Agilent Fragment Analyzer System or Agilent TapeStation. Quantify only the mononucleosomal peak (approximately 75 bp to 250 bp) of the DNA trace to ensure optimal cell-free nucleic acid input.