The QIAseq cfDNA All-in-One workflow consists of two major steps: extraction of cfDNA from plasma and library preparation for NGS.
The QIAseq cfDNA Extraction Kit uses QIAamp Mini Columns to isolate cfDNA from up to 5 ml of plasma. The protocol is specially optimized for NGS with regards to the sensitivity required for variant detection. Following the typical “lyse, bind, wash, elute” steps, cfDNA is first released from histones and selectively bound to the silica membrane. Other contaminants, inhibitors and proteins in the plasma are washed out. In the final step, cfDNA is released from the membrane and eluted in a buffer provided with the kit.
As cfDNA is typically around 170 bp in size, fragmentation prior to library preparation is not required. cfDNA from 1–100 ng in up to 43 µl or 56.5 µl eluant can be directly used for Illumina or Ion Torrent library preparation, respectively.
Library preparation for Illumina sequencers
Library preparation for Illumina sequencers follows a simple one-tube, two-step procedure. In the first step, the ends of the cfDNA are repaired and A-tailed at the 3’ end. This allows the ligation of Illumina-specific sequencing adapters in the second step. The sequencing adapters included with the QIAseq cfDNA All-in-One Kit are dual-barcoded and are provided in a 96-well sealed plate. This allows multiplexing up to 96 different samples in a single sequencing run. Very limited amounts of cfDNA input of less than 10 ng can be amplified using the high-fidelity PCR master mix included with the kit.
Library preparation for Ion Torrent sequencers
Library preparation for Ion Torrent sequencers uses a proprietary All-in-One reaction that combines end-polishing and adapter ligation. End-repair and adapter ligation are performed in one step in a single tube. QIAseq sequencing adapters provided in the kit are dissolved in duplex buffer and are ready to use. Each well of the 24-plex single-use plate contains equimolar mixes of universal and individual barcode adapters. This allows multiplexing up to 24 different samples in a single sequencing run. Only the adapters supplied with the kit are compatible with the innovative All-in-One end repair and ligation reaction. The kit also contains high-fidelity amplification reagents and primers that are specific for Ion Torrent sequencers to enrich for correct adapter-ligated cfDNA molecules.