Accurate next generation sequencing (NGS) is dependent on efficient conversion of input material to sequenceable NGS library molecules. Conversion of degraded input material like that derived from formalin-fixed, paraffin-embedded (FFPE) samples can be challenging. Samples that have been fixed are inherently highly heterogenous due to variation in starting material condition, fixation protocols, storage methods, and sample age. Depending on the sample, the quality of the sample may need to be assessed before library conversion to ensure robust sequencing.
Quality vs quantity
There are 2 main metrics for assessing the quality of DNA derived from FFPE samples: Quality ratio (Q ratio) and DNA Integrity Number (DIN). The Q ratio is determined using qPCR and uses the sizes of DNA fragments to produce a ratio. The DIN uses electrophoresis to measure size distribution and can be measured using a TapeStation™ system (Agilent). A NanoDrop™ Spectrophotometer (ThermoFisher Scientific) or a fluorometer, such as the PicoGreen™ method, can assess the quantity of DNA but does not indicate how much of that DNA can be amplified or converted into NGS libraries.
To find the Q ratio, qPCR is used to amplify DNA fragments of 3 different lengths: short (41 bp), medium (129 bp), and large (305 bp). The short fragments are used to determine the total quantity of DNA present in the sample. The ratios of these fragments indicate the quality. The Q ratio refers to the proportion of 129 bp or 305 bp fragments compared to the 41 bp fragments and is expressed as a fraction: Q129/Q41 and Q305/Q41 respectively. The Q ratio should fall between 0 and 1. If the DNA is degraded into smaller fragments and cross-linked so that it does not amplify well during a PCR reaction, then the number of larger amplicons would be lower, yielding a lower Q ratio. If the FFPE DNA is more intact and does not have damage that interferes with PCR, there will be equivalent amplification of both the larger amplicons and the shorter amplicons, bringing the Q ratio closer to 1.
The more damaged the DNA is, the more difficult it is to amplify larger fragments, so Q305/Q41<0.2 is not an accurate assessment of the DNA quality and cannot be used to accurately predict successful library conversion. For this reason, Q129/Q41 is more commonly used.
qPCR can be an attractive method for assessing DNA quality due to the availability of qPCR machines in most core facilities. Each quality assessment kit uses 5 standards to determine the standard concentration curve and 3 primer sets to assess the DNA quality (1 for each size). Because of the amount of reagents used for the standards, bigger studies are more economical to assess using qPCR. Additionally, each qPCR run uses at least 0.4 ng of DNA. To generate a ratio, the amount is doubled. The amount of starting sample available for your study may limit your DNA QC choices. Lastly, qPCR runs take about 30 minutes.
DNA Integrity Number
The more traditional determiner of DNA quality is the DNA Integrity Number (DIN). After running the Agilent Genomic DNA ScreenTape assay, the DIN is calculated using the TapeStation software. The DIN falls between 1 (highly degraded DNA) and 10 (highly intact DNA). The TapeStation can fit up to 96 samples and requires 5 ng of input sample when analyzing genomic DNA. The assay takes <20 min for 16 samples or <90 min for 96 samples. TapeStation instruments are cheaper than qPCR machines, but the cost per sample to run an assay is comparable to qPCR. DIN is highly correlated with Q ratios (Figure 1), but the correlation is higher for Q129/Q41. Performing QC using the DIN or Q129/Q41 is recommended since Q305/Q41 loses its dynamic range for samples below 0.2.
Figure 1. Quality (Q) ratio is highly correlated with DNA Integrity Number (DIN). Genomic DNA (gDNA) was compared with DNA derived from formalin-fixed, paraffin-embedded (FFPE) samples and formalin-compromised DNA, which mimics FFPE DNA (fcDNA, Horizon). (A) Q129/Q41 is more highly correlated with DIN. (B) Q305/Q41 correlates with DIN above Q305/Q41>0.2 but below that threshold, the Q ratio becomes unreliable.
IDT’s solution for sequencing low-quality DNA
The lower your sample’s quality, the more difficult it becomes to convert it into libraries that can be sequenced using NGS. Since truly assessing DNA damage due to the fixation process is challenging, researchers should not take any risks with their library prep. Having an efficient, single-protocol library prep that works for a range of inputs even without knowing the input quality can save time and money. Use the best possible library kit to ensure robust conversion of input DNA molecules from potentially precious FFPE samples. IDT xGen™ Prism DNA Library Prep Kit can generate quality NGS libraries with as little as 1 ng of input and may allow you to forego DNA QC altogether. The kit’s proprietary ligation strategy maximizes conversion and virtually eliminates adapter-dimer formation. The unique molecular identifier (UMI) sequences incorporated during single-stranded ligation enable a variety of deduplication and error correction strategies. To read about applications of the xGen Prism DNA Library Prep Kit, download our application note, Biomarker discovery—Cancer molecular profiling.