The QIAseq cfDNA All-in-One Kit gives highly uniform exome coverage distributions for cfDNA samples, independent of the input amount. Libraries produced from duplicate 10 ng, 2 ng and 1 ng cfDNA samples were tested.
A. PCR-free library preparation using the QIAseq cfDNA All-in-One Kit was performed using 4 independent replicates of the same circulating cfDNA sample with an input amount of 9.35 ng. Even with inputs of less than 10 ng, the QIAseq cfDNA library prep reagents achieved consistent library yields higher than 2 nM. B. A comparison of normalized coverage distribution for libraries generated from cfDNA samples using the QIAseq cfDNA All-in-One Kit shows no significant differences in coverage for the workflow with or without PCR.
As part of the QIAseq cfDNA All-in-One workflow, circulating cfDNA was isolated using manual QIAamp or automated QIAsymphony protocols. Library prep was performed using 1 ng isolated cfDNA (in 4 replicates), followed by sequencing. A. The QIAseq cfDNA library prep delivers library concentrations that are comparable to those achieved for cfDNA isolated with the QIAamp and QIAsymphony protocols and shows very low sample-to-sample variation. B. Sequencing quality metrics (represented here by the percentage of unique reads) are very good for library preparations from cfDNA isolated with the QIAamp and QIAsymphony protocols as part of the QIAseq cfDNA All-in-One workflow.
Reference samples of cfDNA with known mutations were subjected to the QIAseq cfDNA workflow. Sensitive detection of variants down to even 1% allelic frequency was found. The expected allelic frequencies from Horizon Discovery were used as the standard.
Using either the QIAseq cfDNA All-in-One Kit or a kit from Supplier N, 8 cfDNA samples (input 20–37 ng in a total volume of 43 μl) were used for library construction. The average calculated conversion rate of the replicate samples is displayed. The QIAseq cfDNA All-in-One Kit shows significantly higher conversion rates.
Up to 5 ml plasma can be used for cfDNA extraction using QIAamp Mini Columns and 24 samples are processed in less than 2 h. For library preparation, 1–100 ng of cfDNA can be directly used from up to 56.5 µl eluant volume. Quantification of cfDNA prior to library prep is not necessary. Library prep includes end-polishing and adapter ligation, and takes <1 h. Optional HiFi amplification can be performed for <10 ng cfDNA before samples undergo sequencing.