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.

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.

Features

• 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

Kit Specs

Protocol

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.

KIT CONTENTS

• 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

• Thermocycler

• 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes

• Nuclease-free barrier pipette tips

• Vortex

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.