PreSeq QC Assays

Introduction

Archer® PreSeq® QC assays are a quantitative PCR (qPCR)-based method to determine the quality of the DNA or mRNA in your sample prior to targeted library preparation using the Archer FusionPlex® or VariantPlex® systems.


PreSeq QC assays can be used to pre-screen FFPE samples to determine sample quality prior to NGS library preparation for sequencing on an Illumina® platform. Using a PreSeq assay in the early stage of library prep can save you time and money, maximizing the success rate of the assay by screening for samples that will yield quality libraries for sequencing and analysis.

Highlights

  • Simple qPCR assay shows sample quality prior to NGS library preparation
  • Rescue low quality samples with sample-specific input recommendations
  • Predict expected sensitivity for each sample and limit false negatives

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.


Product information

PreSeq is included in all Archer FusionPlex and VariantPlex kits, and available as an individual order.

AK0067-16: PreSeq DNA QC Assay, 16-reaction kit

AK0043-16: PreSeq RNA QC Assay, 16-reaction kit


Documents

PreSeq DNA protocol PreSeq RNA protocol
PreSeq Calculator PreSeq DNA tech note PreSeq RNA tech note

Kit components

Each kit includes the reagents needed for 16 samples and the primer mix for RNA or DNA quality analysis

PreSeq DNA QC assay reagents:

  • PreSeq DNA QC assay standard
  • PreSeq DNA QC assay 10x primer mix
  • PreSeq RNA QC assay reagents:

  • Step 1: Random priming (x2)
  • Step 2: First strand cDNA synthesis (x2)
  • Step 3: PreSeq RNA QC assay-10x VCP primer mix
  • Step 4: Second strand cDNA synthesis (x2)
  • *The PreSeq RNA QC assay workflow is designed to be performed during library preparation.


    The power of quality scores

    Confidence in NGS data stems from knowledge that the information generated is representative of the sample analyzed. In order to ensure that the sample is accurately represented, data must be gathered prior to library preparation and must have aligning metrics post-sequencing. The Archer PreSeq QC assays enable the user to gather critical information about the sample prior to library preparation that can be tied to sequencing data presented in Archer Analysis. The number of amplifiable genomes in a sample is correlated to mean start sites per gene-specific primer 2 (GSP2), a quality metric in Archer Analysis. The QC score is indicative of sample complexity, and the mean start sites per GSP2 is indicative of library complexity. An ideal QC assay aligns input and library complexity, which increases confidence that data represents the sample.

    Statistical confidence in variant calling is reliant upon the number of unique reads covering the target region. As allele frequency drops due to tumor cellularity or heterogeneity of the biopsy, increasing amounts of unique reads are needed to confidently make a call. The reproducible identification of low allelic frequencies requires the interrogation of hundreds or potentially thousand of unique input molecules. Unfortunately, damage to input molecules has the effect of decreasing the number of sequenceable input molecules contained in a given mass of DNA or RNA. Thus, although 10ng of DNA or RNA theoretically contains ~3,000 copies of every genomic locus, in reality, only a small fraction of those molecules may be available for library preparation. Therefore, mass alone does not help predict sequencing success or library quality.

    Since the QC score generated by PreSeq is dependent on sample quality, it can be used to estimate the number of amplifiable genomes present in a sample. Amplifiable genomes are predictive of the assays sensitivity for mutation detection, including fusions, insertions and deletions (indels) and single nucleotide variants (SNVs), as well as copy number variation (CNV) detection. This information can be used to optimize input based on tumor cellularity, sample quality and sensitivity requirements. Samples that are expected to fail to generate library or fail to meet the labs sensitivity requirements can be withheld from library preparation. Pre-determining poor quality samples can save significant time and cost associated with library prep, sequencing and data analysis.

    Additionally, information gathered from PreSeq can be used in a variety of optimization experiments. Various extraction methods can be benchmarked against each other at little cost to the lab. When used in tandem with Archer Analysis, library preparation and sample handling procedures can be optimized, and incidences of human and sequencing error can be identified. Information on the quality of the sample can also be a critical component of archival sample tracking.


    Formalin fixation and NGS

    Formalin-fixed, paraffin-embedded (FFPE) tissue is one of the most common patient sample types in the clinical research setting, and historical archives represent an enormous biological library for clinical research. Nucleic acid derived from FFPE tissue samples may be utilized in a number of downstream clinical diagnostic test methodologies including next-generation sequencing (NGS), which is becoming a more prominent tool for mutation detection. Unfortunately, the nature of formalin fixation affects target DNA and mRNA molecules and thus the subsequent library preparation and sequence analysis.

    Formalin is a chemical crosslinker that not only crosslinks proteins but also chemically binds RNA to DNA and proteins, thus inhibiting reverse transcription and enabling fragmentation. In addition to the chemical and physical degradation related to the fixation process, storage methods and storage time often cause additional nucleic acid fragmentation.

    Even as clinics transition to NGS to detect gene rearrangements, the technology still relies on the use of historical FFPE tissue samples. While Anchored Multiplex PCR (AMP™)-enabled Archer assays are effective at generating good results from small DNA and RNA fragments, the degradation of archival samples can prevent quality library preparation. Some FFPE samples are of such low integrity that many of the quality control (QC) metrics that are part of the Archer Analysis Pipeline are encumbered. For example, the ability to call fusions using the FusionPlex assay requires fragments at least 60bp long. Determining the sample quality prior to sequencing can help avoid expensive, resource-intensive and time-consuming sequencing failures.

    How to contact us

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    2477 55th Street, Suite 202

    Boulder, CO 80301

    Phone

    Phone: (877) 771 1093

    Phone: (303) 357 9001

    All content © 2018 ArcherDX, Inc.

    For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.