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Next-Gen Sequencing

Cell-Free DNA Library Preparation of Illumina NGS Platforms

ThruPLEX® Plasma-seq

Beckman Coulter Life Sciences the North American distributor for ThruPLEX Plasma-seq. The kit is powered by ThruPLEX chemistry to generate high performance next generation sequencing libraries from cell-free DNA isolated from plasma. The chemistry is optimized specifically for cell-free DNA to maximize the library complexity and to preserve the GC representation of the input DNA. ThruPLEX Plasma-seq libraries can be used directly for whole genome sequencing applications or enriched using a custom panel or the leading target enrichment platforms, including Agilent SureSelect® and Roche NimbleGen SeqCap® EZ.

ThruPLEX Plasma-seq can be completed manually or automated using a Biomek liquid handler depending on your laboratory set up and throughput requirements. If you decide to automate, we have demonstrated methods for ThruPLEX Plasma-seq and other Rubicon Genomics reagent kit.

Overview

Libraries prepared with ThruPLEX Plasma-seq provide added sensitivity to better understand genetic variations found within the plasma samples. Plasma, along with other liquid biopsies, has gained significant interest in the research community as a valuable and easily available sample type. These communities include translational research in oncology and developmental research involving the fetal fraction derived from maternal plasma.

Performance

Highest Diversity and Fewest Unmapped Reads from Cell-Free DNA

ThruPLEX Plasma-seq generated quality libraries with high diversity and a low number of duplicates and unmapped reads. Cell-free DNA was extracted from 3 plasma samples and libraries were prepared at the amounts indicated as measured by Qubit®. The amount of mononucleosomal DNA in each sample, as measured by the Bioanalyzer®, corresponded to 0.09 ng, 0.62 ng, and 15.44 ng. Pooled libraries were sequenced on an Illumina NextSeq® 500 as a paired-end run with 17M to 25M reads per library. Duplication rates were calculated after down-sampling the data to 17 M reads per library.

Reproducible, Unbiased GC Coverage

ThruPLEX Plasma-seq provided the most reproducible and unbiased GC coverage across the human genome. ThruPLEX libraries showed minimal variability across 9 individual plasma samples tested. Libraries were prepared from cell-free DNA isolated from 1 mL of plasma samples and sequenced on an Illumina NextSeq 500. Four separate plasma samples were used to construct the NEBNext® Ultra™ libraries.

Outstanding Target Enrichment Performance

ThruPLEX Plasma-seq libraries were captured at high efficiency and generated data with deep coverage of the kinome for mutation detection. Libraries were prepared from 3 plasma samples at input amounts of 5 ng, 6.5 ng, and 10 ng in triplicate, and targeted sequencing was carried out on an Illumina MiSeq® using samples enriched with the ClearSeq® Human DNA Kinome Panel for SureSelectXT2. On average, 5 M reads were generated per library. Selected bases were successfully captured bases that were in or within 250 bp of the baits.

Advantages

  • Designed for cell-free DNA - Newly formulated repair and ligation reagents
  • High Performance NGS Libraries - High library diversity at low input amount with broad and reproducible GC coverage
  • Adaptable Sample Input Amount - <1 to 30 ng of cell-free DNA
  • Fast and Simple Workflow - 3 steps in a single tube or well in 2 hours with no purification or sample transfer steps
  • Demonstrated method on Biomek FXP Automated Workstation

Webinars & Symposiums

  • Intro to NGS Automation (1 of 3): Simple Automation Solutions. Better Results.

    Click to play  

    Generating NGS data you can trust is highly reliant on the quality of the library sample that’s sequenced.  NGS sample prep has become more streamlined, but it’s still a labor intensive process highly prone to error. Liquid...
  • Intro to NGS Automation (2 of 3) DNA & RNA Sequencing Sample Prep: Automating Simple to Complex Methods

    Click to play  

    DNA and RNA sequencing sample preparation can involve many tedious and error prone steps. Liquid handling automation solutions can help standardize your processes and achieve more consistent results. Beckman Coulter Life Sciences automation and...

  • Intro to NGS Automation (3 of 3) Mission Possible: Automating NGS sample prep for challenging samples and niche applications

    Click to play  

    Accelerating discoveries to improve human and environmental health often require the implementation of niche applications that may involve complex processes and working with challenging samples. 

    During the final...

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    ThruPLEX® Plasma-seq Kit is intended for Research Use Only. It may not be used for any other purposes including, but not limited to, use in diagnostics, forensics, therapeutics, or in humans. ThruPLEX Plasma-seq may not be transferred to third parties, resold, modified for resale or used to manufacture commercial products without prior written approval of Rubicon Genomics, Inc. ThruPLEX Plasma-seq Kit is protected by U.S. Patents 7,803,550; 8,071,312; 8,399,199; 8,728,737 and corresponding foreign patents. Additional patents are pending.
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