PreSeq RNA QC Assay

PreSeq RNA QC Assay

The Archer® PreSeq™ RNA QC Assay is a quantitative PCR (qPCR)-based method to determine the quality of the mRNA in your sample prior to targeted library preparation using the Archer FusionPlex™ system. PreSeq addresses the challenges and importance of measuring the integrity of amplifiable RNA and is predictive of sample sequencing success.


The PreSeq RNA QC Assay can be used to pre-screen FFPE samples to determine sample RNA quality prior to preparing a usable NGS library for sequencing on an Illumina® MiSeq® or Ion Torrent™ PGM™ sequencing platform. Using the 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.

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

PreSeq RNA QC Assay principle

The assay is based on the principle that a control gene of known fragment length can be analyzed by quantitative PCR (qPCR) to determine the relative abundance of that sized fragment in the sample. The assay targets a 113-bp region of the VCP gene, which is ubiquitously expressed housekeeping gene.



Easy Workflow

The PreSeq workflow is designed as a series of sequential transfers of input nucleic acid into tubes that contain all of the required enzymes and buffers required for double-stranded cDNA synthesis.

  1. Random priming - random hexamers hybridize to the RNA to generate random start sites for cDNA synthesis, maximizing sample coverage
  2. First strand synthesis - reverse transcriptase binds to the primed RNA and synthesizes the first cDNA strands
  3. PreSeq™ RNA QC assay - analyze a small aliquot of the cDNA by qPCR to determine input sample quality
  4. Second strand synthesis – while the cDNA is being analyzed, the remaining RNA in the sample is digested, and polymerase synthesizes the second cDNA mixture. The resulting double-stranded cDNA product is stable, allowing the user to obtain qPCR results prior to deciding which samples should be brought through the remainder of library preparation
PreSeq RNA QC Assay workflow

The PreSeq RNA QC Assay workflow is designed for use with minimal input, so quality control can be performed during library preparation instead of sacrificing more precious sample for separate RNA analysis.


PreSeq™ Kit Components

The PreSeq™ RNA QC Assay kit includes the reagents needed to prepare double-stranded cDNA from 16 samples and the primer mix for RNA quality analysis.

Archer 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)

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 variant detection. Unfortunately, the nature of formalin fixation affects target 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 rendering RNA susceptible to 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.


RNA integrity affects library preparation

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 FusionPlex Assays are effective at generating good results from small RNA fragments, the degradation of archival RNA 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 RNA quality prior to sequencing can help avoid expensive, resource-intensive and time-consuming sequencing failures.


Measuring RNA quality

There are various methods to determine RNA quantity, including

MethodLimitation
UV absorbance Does not determine fragment length or degree of RNA crosslinking
Fluorescent dye-based quantification Does not determine fragment length or degree of RNA cross linking
Gel electrophoresis Does not provide an quantifiable integrity score

RNA quantity by itself is insufficient for predicting successful fusion calling; the RNA fragment length is actually more important. It is therefore important to select an RNA QC assay that supports the intended downstream application. RNA integrity, or mean amplicon length, is a more applicable metric for predicting NGS assay success than RNA quantity. Currently, many researchers rely on an RNA integrity number (RIN) score generated by Agilent BioAnalyzer™ cDNA traces to predict sample RNA quality, However, RIN scores fail to provide accurate assessment of the quality of RNA necessary for library preparation. RIN scores are based on a ratio of the 5kb 28S and 2kb 18S rRNA species present in a sample, but they rely on the assumption that the quality score generated from the abundance of rRNA species (>80% of RNA) is suggestive of the quality of the underlying 1-3% mRNA population used in sequencing. The mRNA in a cell is turned over much more rapidly than the stable rRNA as the cell continuously adjusts for specific protein requirements; therefore a 28S:18S ratio may not be representative of the mean mRNA fragment size in a sample.

In order to address the inconsistency of RNA quality from FFPE and the lack of accurate RNA quality analytical methods suited for NGS applications, we developed the Archer PreSeq RNA QC Assay to predict the success of library preparation using FusionPlex Assays.


Related Topics

How to contact us

Address

2477 55th Street, Suite 202

Boulder, CO 80301

Phone

Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2017 ArcherDX, Inc.

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