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)](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/cell-free-dna/qiaamp-minelute-ccfdna-kits) - [MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher)](https://www.thermofisher.com/order/catalog/product/A29319): the manual isolation method was used from the MagMAX protocol. - [Apostle MiniMax ™ Isolation Kit (Beckman Coulter)](https://www.mybeckman.uk/reagents/genomic/dna-isolation/from-plasma/c40603) - [nRichDX ](https://www.nrichdx.com/cfdna) 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](/document/ligation-sequencing-v14-human-cfdna-singleplex-sqk-lsk114). 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). 
| Extraction kit | Replicate | Plasma volume (ml) | Extraction yield (ng) | Library yield (ng) | Output (Gb) | Read count (M) | Read N50 (bases) | | --- | --- | --- | --- | --- | --- | --- | --- | | QIAamp MinElute ccfDNA Kit (QIAGEN) | 1

2 | 4

4 | 17.4

16.5 | 7.7

8.6 | 70.1

65.6 | 265

246 | 224

226 | | MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher) | 1

2 | 4

4 | 13.4

12.1 | 4.2

5.4 | 44

56 | 172

220 | 222

225 | | Apostle MiniMax Isolation Kit (Beckman Coulter) | 1

2 | 4

4 | 16.3

13.3 | 9.0

6.4 | 59.8

81.3 | 237

328 | 220

227 | | nRichDX | 1

2 | 4

4 | 15.3

17.1 | 6.7

8.6 | 76.8

67.6 | 275

240 | 234

238 | __Table 1.__ Library preparation and sequencing statistics for cfDNA libraries prepared in this study.
![cfDNA Extraction Kit Know-how-image1](https://a.storyblok.com/f/196663/edd6985bd7/extraction_kit_know-how-image1-5zph14bpcx8uzokaeiyy3q-ee84a026042c6587707e4d5b0c3c75b4.svg) __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.

Date	Version	Language	Device		Changes
6/6/2025	-	English	-		-
8/7/2024	-	English	-		-

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