Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation.
Author(s): Dang J, Mendez P, Lee S, Kim JW, Yoon JH, Kim TW, Sailey CJ, Jablons DM, Kim IJ
Publication: Int J Oncol, 2016, Vol. 49, Page 1755-65
PubMed ID: 27511764 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of using different methods of DNA quantification and extraction on next generation sequencing (NGS) library yields and success. DNA yields from frozen specimens were compared with those from FFPE specimens.
Conclusion of Paper
Measurement of DNA concentrations differed among the quantification methods. NGS library yields from high quality specimens were comparable between the in-house TaqMan and RNAse P quantification methods, but library yields from low quality specimens were higher when quantified using TaqMan RNase P assay. Quantification based on the shorter amplicon of the in-house SYBR green-based assay resulted in higher yields than use of the longer amplicon or the TaqMan RNase P assay. It was possible to obtain sequencing data from low quality specimens using the dual amplicon SYBR green-based quantification but not by using other methods that did not account for degradation.
Studies
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Study Purpose
This study compared the effects of using different methods of DNA quantification on next generation sequencing (NGS) library yields and success. DNA was extracted from 5-10 µm sections of 45 FFPE blocks containing tumor (one GI/pancreaticobiliary adenocarcinoma and the rest unspecified) using the UltraRapid FFPE DNA Extraction kit following deparaffinization in xylene. DNA was quantified using Quant-iT PicoGreen dsDNA Assay kit, Qubit dsDNA HS assay kit, an in-house TaqMan qPCR assay, TaqMan Copy Number Reference Assay for human RNase P, and an in-house SYBR-Green assay. Primers for the in-house qPCR methods were designed in regions of the genome without polymorphic sites or copy number variation. The in-house TaqMan assay included a small (≤96 bp) and large (<190 bp) amplicon while the in-house SYBR assay included a pool of two small (≤85 bp) amplicons and a pool of two large (<190 bp) amplicons. NGS libraries were prepared from 10 ng DNA using the CureSeq NextDay Seq-Pan Cancer HotSpot Panel kit and yield was determined by a bioanalyzer.
Summary of Findings:
The DNA concentrations as determined by TaqMan RNase P were higher than by PicoGreen (average 1.4 to 1.6-fold) or Qubit fluorometry (average 2.88-fold), and as determined by the longer amplicons of the in-house TaqMan quality control assay (1.62-fold) and SYBR green assays (1.70-fold), but lower than when determined using the shorter amplicon for the in-house TaqMan quality control assay or the SYBR assay. Concentrations by TaqMan RNase P were weakly correlated with those by PicoGreen (R2=0.28-0.37) and the longer amplicon for the in-house TaqMan quality control assay (R2=0.36), modestly correlated with those by the longer and shorter amplicons in the SYBR assay (R2=0.55 and R2=0.69, respectively), but strongly correlated with those using the small amplicon in-house TaqMan assay (R2=0.86). When compared to the in-house SYBR assay, the in-house TaqMan assays reported higher yields both for the small amplicons (1.29-fold) and the larger ones (1.18-fold), and concentrations were strongly correlated for the small amplicons (R2=0.89) and very strongly correlated for the larger amplicons (R2=0.90).
Overall, DNA found to be of high quality using the in-house TaqMan assay (ratio of short to long amplicons of 0.95 to 1.50) produced comparable average yields using the TaqMan RNase P and the in-house TaqMan assay for quantification (10,009.90 pM versus 9,922.60 pM), but absolute library yields were higher for 3 of 4 specimens when quantified using the in-house TaqMan assay. For poor integrity DNA (ratio of short to long amplicons of >4), average library yields were higher when made based on the TaqMan RNase P assay than the in-house TaqMan assay (3624.00 pM versus 1639.56 pM). Using the large amplicon in the SYBR green based assay for quantification, resulted in lower library yields than use of the smaller amplicon (1773.60 pM versus 3609.47 pM), but quantification based on the small amplicon in the SYBR green-based assay resulted in higher library yields than RNAse P (3609.47 pM versus 1196.51 pM). Importantly, it was possible to obtain sequencing data from low quality specimens using the SYBR green-based quantification but not by using other methods.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Automated electrophoresis/Bioanalyzer DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Technology platform Quant-iT PicoGreen dsDNA Assay kit
Qubit dsDNA HS assay kit
In-house TaqMan qPCR assay
TaqMan Copy Number Reference Assay for human RNase P
In-house SYBR-Green assay
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Study Purpose
This study compared DNA yields from FFPE and frozen specimens and investigated the differences between DNA extraction methods. DNA was extracted from 5-10 µm sections of 45 FFPE blocks containing tumor (type and processing unspecified) using the UltraRapid FFPE DNA Extraction kit following deparaffinization in xylene. For five specimens, DNA was also extracted using the AllPrep DNA/RNA FFPE kit. DNA was extracted from 26 frozen specimens using the DNeasy Blood & Tissue kit with the QIAamp kit. DNA was quantified using Quant-iT PicoGreen dsDNA Assay kit, Qubit dsDNA HS assay kit, an in-house TaqMan qPCR assay, TaqMan Copy Number Reference Assay for human RNase P, and an in-house SYBR-Green assay. Primers for the in-house qPCR methods were designed in regions of the genome without polymorphic sites or copy number variation. The in-house TaqMan assay included a small (≤96 bp) and large (<190 bp) amplicon while the in-house SYBR assay included a pool of two small (≤85 bp) amplicons and a pool of two large (<190 bp) amplicons.
Summary of Findings:
The DNA yields were higher from frozen lung tumors than FFPE specimens (21.59 ng/µL versus 1.15 ng/µL). Further, the D-scores (ratio of short to long amplicons) were higher for FFPE than frozen specimens (2.34 versus 1.07), indicating DNA degradation. DNA yields were comparable when extracted with the column-based AllPrep DNA/RNA FFPE kit and the UltraRapid FFPE DNA Extraction kit (4.94 ng/µL and 4.58 ng/µL, respectively). Further, the D-score was unaffected by extraction method (1.48 versus 1.18).
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Automated electrophoresis/Bioanalyzer 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
UltraRapid FFPE DNA Extraction kit
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen