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Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

Author(s): Kresse SH, Namløs HM, Lorenz S, Berner JM, Myklebost O, Bjerkehagen B, Meza-Zepeda LA

Publication: PLoS One, 2018, Vol. 13, Page e0197456

PubMed ID: 29771965 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of extraction protocol on the yield, integrity, amplificability, and NGS metrics of DNA and RNA from FFPE sarcoma specimens. Five archival FFPE blocks (2-5 years old) of sarcomas were obtained from pathology archives. The blocks included an alveolar soft tissue sarcoma, an alveolar rhabdomyosarcoma, a synovial sarcoma with a known gene fusion, an extraskeletal myxoid chondrosarcoma with a known gene fusion, and a Ewing sarcoma with a known gene fusion. No details of specimen procurement, processing, or storage were provided but the authors state they were obtained using standard procedures. DNA was extracted from a freshly cut 10 µm section using the AllPrep DNA/RNA FFPE Kit, GeneRead DNA FFPE Kit, QIAamp DNA FFPE Tissue Kit, or the truXTRAC FFPE DNA Kit. RNA was extracted from a freshly cut 10 µm section using a modification of the AllPrep DNA/RNA FFPE Kit, RNeasy FFPE Kit, using a modification of the Agencourt FormaPure Kit, or truXTRAC FFPE RNA Kit. DNA and RNA extractions were performed simultaneously and separately using the AllPrep DNA/RNA FFPE Kit and only simultaneously using the truXTRAC FFPE kit. Two to three separate extractions were preformed using each kit. DNA and RNA yield were determined using the Qubit dsDNA and RNA Assay kits. DNA integrity was evaluated using a TapeStation and RNA using a bioanalyzer. DNA and RNA amplificability were investigated using the Illumina FFPE QC kit and the ArcherDX PreSeq QC kit, respectively. SureSelect XT DNA libraries from four specimens were pair-end sequenced on a HiSeq4000. Sequencing libraries were prepared from RNA from the three specimens with known gene fusions using the Archer FusionPlex Sarcoma Assay and the Archer Universal RNA Reagent Kit v2 and sequenced using the MiSeq system.

   Summary of Findings:

   DNA yields were highest when extraction was performed simultaneously with RNA extraction using the truXTRAC DNA/RNA kit and yields using this kit were significantly higher than when extraction was with the QIAamp, GeneRead and AllPrep kits (P<0.05) and non-significantly higher than when performed using the same kit not in combination with RNA extraction. DNA integrity numbers were significantly higher using the truXTRAC DNA/RNA kit simultaneously for RNA extraction than for any of the other kits, with the exception of the AllPrep kit. Delta CT values using the simultaneous protocol with the truXTRAC DNA/RNA protocol were lower than using the GeneRead, QIAamp, and AllPrep kits (P=0.04). One AllPrep and one GeneRead extraction did not yield sufficient DNA for library preparation; however, the yield and library metrics were not significantly different among extraction methods for the remaining samples. The majority of SNVs called with either pipeline (Mutect or Strelka) were concordant. A trend toward a lower duplication rate was observed for specimens extracted using AllPrep.

   Differences between RNA extraction kits were smaller than for DNA but the highest yields were obtained using the truXTRAC DNA/RNA kit for RNA alone or in combination with DNA. Using the truXTRAC DNA/RNA kit, yields were significantly higher than with the FormaPure kit (P=0.043). RNA integrity numbers were significantly higher using the truXTRAC RNA than either the RNeasy or FormaPure kit (P=0.04). RNA amplificability as determined using the PreSeq QC assay were unaffected by extraction method. Sequencing library yields were highest for RNA extracted using the FormaPure and truXTRAC DNA/RNA kits, but the differences were non-significant. The fusion was detected regardless of extraction method for one specimen, detected only when extraction was with FormaPure or truXTRAC in another specimen, and the RNA was of insufficient quality to allow for detection in the third specimen, regardless of extraction method. When the data was down-sampled such that all specimens were a comparable number of reads, the highest percentage of unique reads and on-target reads was obtained in specimens extracted using the FormaPure kit followed by the truXTRAC kit, but the differences were not significant.

   Biospecimens

   • Tissue - Not specified

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Next generation sequencing
   DNA	Fluorometry
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Fluorometry
   RNA	Next generation sequencing
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	AllPrep DNA/RNA FFPE Kit GeneRead DNA FFPE Kit QIAamp DNA FFPE Tissue Kit truXTRAC FFPE DNA Kit RNeasy FFPE Kit A modification of the Agencourt FormaPure Kit truXTRAC FFPE RNA Kit truXTRAC FFPE DNA/RNA Kit
   Next generation sequencing Specific	Data handling	Strelka Mutect

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