Rapid Cell Free DNA Library Prep for Sequencing on Ion Torrent™ Platforms

The NEXTFLEX® Cell Free DNA Sequencing Kit for Ion Torrent™ platforms is designed to prepare genomic DNA-Seq NGS libraries from 1 – 5 ng of cell free DNA for sequencing using the, Ion S5™, Ion S5™ XL, Ion Torrent™ PGM, and Ion Proton™ sequencing platforms.

The NEXTFLEX® Cell Free DNA Sequencing Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time-consuming steps in library preparation. In addition, the availability of up to 64 unique adapter barcodes enables high-throughput applications.

There are five main steps involved in preparing genomic DNA for Ion Torrent sequencing: DNA extraction, DNA end repair, size selection (optional), adapter ligation and PCR amplification. This kit contains the necessary material to prepare the user’s purified DNA for emulsion PCR and subsequent sequencing. We recommend using the NextPrep-Mag™ cfDNA Isolation Kit for isolation of circulating DNA from blood plasma prior to library prep.

Cell free DNA has become a powerful marker in clinical applications due to the unique origin of DNA molecules present in plasma. Detection of fetal DNA from maternal plasma has proven to be a viable, non-invasive option to identify a variety of fetal traits including: sex determination, sex chromosome-linked disorders and aneuploidy events. Circulating tumor DNA extracted from plasma of symptomatic patients can be used as a non-invasive resource for diagnosis, prognosis, treatment decisions, and follow-up monitoring of cancer patients.

Cell Free DNA-Seq Multiplexing Options

The NEXTFLEX® DNA Barcodes for Ion Torrent™ platforms are adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. This format enables multiplexing of up to 64 samples. The NEXTFLEX® DNA Barcodes are available in sets of 8, 16, 32, and 64 unique adapters. The ability to pool samples in an efficient way significantly decreases hands-on time while providing robust data quality.

Features

• Only 1 – 5 ng of cell free DNA input required

• Enhanced Adapter Ligation Technology offers larger number of unique sequencing reads

• Flexible barcode options– Kits contain 8, 16, 32, or 64 unique barcodes

• Simplified workflow with 96-well plate compatible volumes

• Ideal for Ion Torrent™ DNA-Seq library prep from cell free fluids

  
  

Kit Specs

NEXTflex™ Cell Free DNA-Seq Protocol

KIT CONTENTS

• NEXTFLEX® End Repair Buffer Mix

• NEXTFLEX® End Repair Enzyme Mix

• NEXTFLEX® Ligation Mix

• NEXTFLEX® Adapter Mix (6.25 µM)

• NEXTFLEX® Primer Mix (12.5 µM)

• NEXTFLEX® 5X PCR Master Mix

• Nuclease-free Water

• Resuspension Buffer

REQUIRED MATERIALS NOT PROVIDED

• 1 – 5 ng of cell free DNA in up to 40 µL nuclease-free water

• NEXTFLEX® DNA Barcodes for Ion Torrent™ Platforms – 8 / 16 / 32 / 64 (Cat # 4010-01, 4010-02, 4010-03, 4010-04)

• 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 5 mL (Beckman Coulter Genomics, Cat # A63880)

• Magnetic Stand -96 (Ambion, Cat # AM10027) or similar

• Thermocycler

• 2, 10, 20, 200 and 1000 µL pipettes or multichannel pipettes

• Nuclease-free barrier pipette tips

• Microcentrifuge

• 1.5 mL nuclease-free microcentrifuge tubes

• Vortex