For Use with Spotlight Panels
For next-generation sequencing (NGS), quantity and quality of circulating cell-free DNA (cfDNA) is essential to good library preparation. We recommend the following methods for extraction and quantification of cfDNA.
Input quantification by spectrophotometric-based (NanoDrop®) or Fluorometric-based (Qubit®)
methods may not provide an accurate assessment of the usable DNA within the sample.
Quantification by spectrophotometric-based methods commonly overestimates DNA concentration and is limited to relatively high concentration samples. Quantification by Fluorometric-based methods provides accurate DNA concentrations for samples with high quality DNA (e.g., whole blood, fresh frozen samples, cultured cells), but performs poorly with challenging samples and cannot distinguish between circulating, cell-free DNA (cfDNA) and high molecular weight cellular gDNA. Therefore, for challenging samples such as cfDNA, we recommend quantification by a qPCR-method, using a short amplicon to accurately determine the concentration sample DNA [Simbolo M. et al. PLoS ONE (2013) 8(6): e62692].
Alu sequences (highly abundant in the human genome) can be used for the sensitive quantification of human genomic DNA. For qPCR-based determination of sample quantity and integrity, xGen Input DNA Quant Primers (Alu primers) are avaiable. Following input analysis, the appropriate amount of sample DNA can be used as input for NGS library preparation.
Circulating cfDNA Samples
Cell-Free DNA BCT® tubes (Streck Cat. No. 218961) and the QIAamp Circulating Nucleic Acid Kit (Qiagen Cat. No. 55114) or Quick-cfDNA Serum & Plasma Kit (Zymo Research Cat. No D4076) are recommended for sample collection and cDNA extraction. Quantification by qPCR is recommended to determine the concentration of the input cfDNA.
As cfDNA exhibits a narrow size distribution around 165 bp, Alu115-qPCR results accurately detect the total quantity of cfDNA and high molecular weight cellular gDNA.
Before You Start
Required Materials Not Supplied
For cfDNA extraction
- Cell-Free DNA BCT® tubes (Streck Cat. No. 218961) or BD Vacutainer® Plus Plastic K2EDTA tubes (BD Cat. No 368589)
- QIAamp Circulating Nucleic Acid Kit (Qiagen Cat. No. 55114) or Quick-cfDNA Serum & Plasma Kit (Zymo Research Cat. No D4076)
- Microfuge tubes (1.5 and 2.0mL tubes)
- 15mL conical tubes
- Serological pipets
- Aerosol-resistant tips and pipette ranges from 1-1000uL
- Dry ice
- xGen Input DNA Quant Primers (IDT Cat. No. 10009856)
- iTaq™ Universal SYBR® Green Supermix (Bio-Rad Cat. No. 172-5120)
- Standard human genomic DNA (Promega Cat. No. G3041)
- Programmable thermocycler operating within manufacturer's specifications (Bio-Rad CFX96)
- 0.2mL PCR tubes or 96-well plate
- Aerosol-resistant tips and pipette ranges from 1-1000uL
- Nuclease-free water (molecular biology-grade) or low EDTA TE buffer
- Bioanalyzer, Tape Station, Fragment Analyzer or similar technique to assess the quality and size distribution of cfDNA (http://www.agilent.com/cs/library/usermanuals/Public/4200 TapeStation_HS-D1000_QG.pdf)
The Input DNA Quant Primers Kit contains enough Alu115 primer pairs for the preparation of 96 reactions.
- Draw blood in K2EDTA tube and process it IMMEDIATELY for plasma isolation. Blood must be processed within 2 hours from the time of collection. Avoid unnecessary agitation.
- Alternatively, blood may be drawn into Streck's Cell-Free DNA BCT® tubes. Proceed to plasma isolation as soon as possible.
- Separate plasma by centrifugation at 3000 x g for 10 minutes. Spin down only one time.
Avoid touching or disturbing the buffy coat layer. Leaving a small amount of plasma above the buffy coat layer will help minimize genomic DNA contamination.
- Store plasma in appropriate aliquots. Place on dry ice and store at -80°C or immediately proceed to cfDNA extraction. cfDNA may be extracted from plasma stored at -80°C for several months or even years later.
- For samples collected in K2EDTA tubes, extract cfDNA following exact protocols from
- For sample collected in Streck's Cell-Free DNA BCT® tubes, follow Streck's recommendation during Proteinase K treatment of increasing incubation at 60 C for 1 hour (https://www.streck.com/wp-content/uploads/sync/Collection/Collection_Tubes/Cell-
- Proceed to cfDNA quantification.
cfDNA Quantification using Alu Assay
- Prepare a standard curve using serial dilutions of human genomic DNA of known quantities
(10, 1, 0.1, 0.01, 0.001ng) for each Alu primer pair in duplicate.
- Prepare to run each sample and a no template control in duplicate for sample quantification. Determine the volume of sample DNA to load so as to increase the likelihood it will fall within the standards and, therefore, the dynamic range of the assay. For limiting samples, a minimum of 1 ul is required. If your DNA is more concentrated than the highest standard, dilute it to fall between the standards.
- Prepare the qPCR reaction in a 1.5mL tube by adding reagents in the order listed below. We
suggest the use of iTaq Universal SYBR Green Supermix (Bio-Rad 172-5120).
- Place in the thermocycler and run the Alu Primer PCR Quantification program as described below.
- Plot Ct values (y-axis) VS. DNA quantity of the serial dilutions (x-axis) on a log scale to
produce the standard curve. Identify the slope and the y-intercept. Solve using the following
formula to determine the sample DNA concentration.
- The concentration for the Alu115 amplicon can be used to determine the total quantity of
usable DNA in ng/L. Verify that the calculated concentration of your sample is between the
DNA standards of the assay. The minimum concentration is 1 ng/L (4ng/uL recommended).
The minimum input quantity is 5ng per reaction (minimum 10ng minimum recommended).
- Use Bioanalyzer, Tape station, Fragment Analyzer, or similar technique to assess the quality
and purity of cfDNA (http://www.agilent.com/cs/library/usermanuals/Public/4200-
TapeStation_HS-D1000_QG.pdf). Examples of appropriate traces are shown below. While
not all cfDNA samples have identical size distributions, it is critical that the trace shows a
predominant mononucleosomal cfDNA peak at approximately 165 base pairs.
- Typical yield of healthy donor blood is 2 to 3 ng/mL plasma and of tumor patients is