IDEAL FOR SEQUENCING OF cfDNA & ctDNA ON AN ILLUMINA® SEQUENCING PLATFORM
NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0 for Illumina® Sequencing Platforms
- Designed for low sample input – 10 ng of input cfDNA required
- Accelerated workflow with minimal hands-on time
- Flexible adapter barcode options – up to 1,536 unique dual index barcodes are available
- Compatible with Illumina® sequencing platforms
- Protocols available for automation on the Sciclone® G3 NGSx, NGSx iQ™ and Zephyr® G3 NGS workstations
Prepare Libraries in Only 2 Hours
The NEXTFLEX® Cell Free DNA-Seq Kit 2.0 can produce libraries from 10 ng of cell free DNA for circulating tumor DNA (ctDNA) or cell free fetal DNA (cffDNA) analysis, in two hours. This low-input library preparation kit delivers high coverage quality and reduced bias for Illumina® sequencing applications. The NEXTFLEX® Cell Free DNA-Seq Kit 2.0 can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina®platforms. The availability of up to 1,536 unique dual index barcodes facilitates high-throughput applications without compromising data integrity.
We recommend using the NextPrep-Mag™ cfDNA Automated Isolation Kit for isolation of circulating cfDNA from blood plasma prior to library prep with the NEXTFLEX® Cell Free DNA-Seq Kit 2.0.
Reduced Bias and Coverage Gaps
NEXTFLEX® Cell Free DNA-Seq Kit 2.0 exhibits minimal GC bias and produces uniform coverage of difficult to sequence genome regions. This kit also offers robust ligation efficiency, resulting in greater library diversity and a larger number of unique sequencing reads. These optimizations enable investigators to obtain more information from each sequencing run.
Flexible Multiplexing Options
The NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0 for Illumina® sequencing platforms is designed to be used with the NEXTFLEX® Unique Dual Index barcodes (384 barcodes) or the NEXTFLEX® Unique Dual Index barcodes (1,536 barcodes). Thesed NEXTFLEX® Adapters are long, annealed adapters that offer an improved multiplexing workflow and flexible setup.
This kit is automated on the Sciclone® G3 NGSx, NGSx iQ™ and Zephyr® G3 NGS workstations. Contact us at [email protected] for more details.
- NEXTFLEX® cfDNA Enrichment Beads
- NEXTFLEX® End-Repair & Adenylation Buffer Mix 2.0
- NEXTFLEX® End-Repair & Adenylation Enzyme Mix 2.0
- NEXTFLEX® Ligase Buffer Mix 2.0
- NEXTFLEX® Ligase Enzyme 2.0
- NEXTFLEX® PCR Master Mix 2.0
- NEXTFLEX® Primer Mix 2.0 (50 μM)
- Nuclease-free Water
- NEXTFLEX® Resuspension Buffer
- NEXTFLEX® Cleanup Beads 2.0 1.5 mL
REQUIRED MATERIALS NOT PROVIDED
- ~1 ng of Cell-Free DNA in up to 32 µL nuclease-free water.
- NEXTFLEX® DNA Barcodes – 6 / 12 / 24 / 48 (Cat # 514101, 514102, 514103, 514104) or NEXTFLEX-96™ DNA Barcodes (Cat # 514106)
- Ethanol 100% (room temperature)
- Ethanol 80% (room temperature)
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
- Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
- Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar
- 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
Figure 1. Nucleosome enriched libraries
10 ng input of cfDNA was used for the enrichment (or no enrichment) and prepared using the NEXTFLEX® cell-free DNA-seq kit 2.0 standard protocol with the following enrichments.
- A) No enrichment
- B) Mono-nucleosome enrichment
- C) Mono-, di-, tri-nucleosome enrichment
Figure 2. Automated NEXTFLEX® cell-free DNA-seq kit 2.0 libraries
cfDNA was isolated from 5 mL of plasma using the NextPrep-Mag™ cfDNA automated isolation kit on the chemagic™ 360 instrument and cleaned/concentrated to be eluted in 32 µL into the NEXTFLEX® cell-free DNA-seq kit 2.0 using 9 cycles of PCR (without nucleosome enrichment. Libraries were diluted 1:3 and run on the LabChip® GXII Touch™ HT nucleic acid analyzer.
Papers that Cite the Use of the NEXTFLEX® Cell-Free DNA-Seq Kit:
- Xia, S. et al. (2015) Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls. Lung Cancer. doi: 10.1016/j.lungcan.2015.07.002.
- High-Throughput, End-to-End Cell Free DNA Analysis Workflow from Plasma
- A Start-to-Finish cfDNA Extraction & Sequencing Automation Experience from Plasma/Serum
Adapters for multiplexing
- NEXTFLEX® Unique Dual Index Barcodes
- NEXTFLEX-HT™ Barcodes
- NEXTFLEX® DNA Barcodes
- NEXTFLEX-96™ DNA Barcodes
- NEXTFLEX® Dual-Indexed DNA Barcodes
Kit for cfDNA Isolation