IDEAL FOR SEQUENCING OF cfDNA & ctDNA ON AN ILLUMINA® SEQUENCER
NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0 for Illumina® Sequencing
- Designed for low sample input – 10 ng of input cfDNA required
- Accelerated workflow with minimal hands-on time
- Flexible adapter barcode options – up to 384 single index or unique dual index barcodes are available
- Compatible with Illumina® sequencing platforms
- Protocols available for automation on the PerkinElmer® Sciclone® and Zephyr® G3 NGS workstations
For research use only. Not for use in diagnostic procedures.
COMPATIBLE DNA BARCODES
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 384 single-index single index and 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 is designed to be used with the NEXTFLEX® Unique Dual Index barcodes, NEXTFLEX® DNA barcodes, NEXTFLEX-96™ DNA barcodes and NEXTFLEX-HT™ barcodes. These barcodes are available in sets of 6, 12, 24, 48, 96 and 384 unique indexed adapters. All of these NEXTFLEX® Adapters are long, annealed adapters that offer an improved multiplexing workflow and flexible setup.
- 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)
- Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63880)
- 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.
- Optimized Library Prep for Cell-Free DNA from Human Plasma