Circulating cell-free DNA (cfDNA) serves as a non-invasive biomarker within plasma, providing insights into the genetic characteristics of various diseases. Our recommended method for extracting cfDNA from blood/plasma samples uses the QIAamp MinElute ccfDNA Kit (QIAGEN). In this study we assess the performance of other commonly used cfDNA extraction kits with regards to cfDNA yield and sequencing performance.

Methods and Results

A bulk plasma sample was collected from a single donor, stored in K2 EDTA and delivered overnight at 4°C to our facility. On the day of receipt, the plasma was centrifuged at 16,000 x g for 10 minutes to remove residual cells and aliquoted into duplicate 4 ml samples per extraction kit.

cfDNA was extracted according to manufacturers’ instructions for each extraction kit:

• QIAamp MinElute ccfDNA Kit (QIAGEN)
• MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher): the manual isolation method was used from the MagMAX protocol.
• Apostle MiniMax ™ Isolation Kit (Beckman Coulter)
• nRichDX

The extracted cfDNA was quantified using a Qubit dsDNA HS Kit (Thermo Fisher) and fragment length assessed using the Femto Pulse (Agilent) (Table 1 and Figure 1A). Library preparation was then performed following the Ligation Sequencing v14 – Human cfDNA Singleplex protocol. Libraries were sequenced on PromethION R10.4.1 Flow Cells for 72 hours, setting the minimum read length to 20 bp. Read lengths and flow cell outputs are documented (Table 1 and Figure 1B).

Table 1. Library preparation and sequencing statistics for cfDNA libraries prepared in this study.

Figure 1. Length profiles of cfDNA extracted from four different commonly used kits. A) Fragment length profiles determined using the Femto Pulse system (Agilent). B) Sequencing read length profiles generated on PromethION, showing the characteristic nucleosomal peak patterns of cfDNA libraries.

Summary

This study assesses four different cell-free DNA (cfDNA) extraction kits for their performance with nanopore sequencing. We observed the highest extraction yields with the currently recommended QIAamp MinElute kit, though yields using Apostle and nRichDX kits were closely comparable. The MagMAX kit (following the manual extraction protocol) had the lowest yields, though it should be noted that this is primarily designed for extraction using a KingFisher instrument. Sequencing performances all fell into acceptable ranges for output, read count and N50s except for the MagMAX kit which had lower outputs (likely due to lower ng inputs loaded on the flow cells). While our recommendation is to use the QIAamp MinElute ccfDNA Kit, users that have access to other extraction kits could expect to observe similar performance.

Change log

Version	Change
v1, August 2024	Initial publication