DNA Sequencing

DNA sample QC for NGS

First, it is necessary to evaluate the quality of the genomic DNA (integrity and purity).

The integrity and size of genomic DNA can be checked by regular or pulse-field gel electrophoresis (PFGE) using an agarose gel. Regular gel electrophoresis is not highly accurate, since large DNA molecules migrating through a gel will essentially move together in a size-independent manner. However, it will provide sufficient information in terms of integrity (size range) and purity (RNA contamination runs as a diffuse smear at the bottom of the gel). So, it is still one of the most popular methods for accessing genomic DNA quality.

Note: RNA contamination can lead to overestimation of DNA concentration and may inhibit some downstream steps. When RNA contamination is evident, treat the sample with DNase-free RNase I.

The ratio of the readings at 260 nm and 280 nm (A260 / A280) on a spectrophotometer provides an estimate of DNA purity with respect to contaminants that absorb UV light, such as protein. Pure DNA has an A260/ A280 ratio of 1.7–1.9.

Note: For accurate A260 /A280 values, measure absorbance in slightly alkaline buffer (e.g., 10 mM Tris•Cl, pH 7.5).

The second step is to measure the concentration of genomic DNA.

DNA concentration can be determined by measuring the absorbance at 260 nm in a spectrophotometer. The Nanodrop instrument is becoming widely used, owing to its low sample volume (1 µl) and convenience (no cuvettes required). To ensure reliability, readings should be between 0.1 and 1.0.

Note: The absorbance measurements cannot discriminate between DNA and RNA and RNA contamination can lead to overestimation of DNA concentration. However, the A260 / A280 ratios are different with pure RNA reading ~2.0 and pure DNA reading ~1.8. Therefore, a reading of, for example, 1.95 can suggest RNA contamination.

Note: Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, due to an upward shift in the A260 value.

Fluorometry allows specific and sensitive measurement of DNA concentration by use of the fluorochromes. In addition to Hoechst 33258, which shows increased emission at 458 nm when bound to DNA, more sensitive fluorochromes, such as PicoGreen dye, are now used. PicoGreen dye-based assays are up to 10,000-fold more sensitive than UV absorbance detection and at least 400-fold more sensitive than assays that use the Hoechst 33258 dye. Unlike UV absorbance, PicoGreen assays are highly selective for dsDNA over RNA and ssDNA.

DNA standards and samples are mixed with fluorochrome and measured on a fluorometry instrument. The sample measurements are then compared to the standards to determine DNA concentration.

Real-time PCR assays can be used to assess quantity and quality of DNA samples. Multiplex PCR assays that use primer sets that amplify fragments of different size at multiple loci can provide an effective quality control for identifying damage or fragmentation. These assays specifically measure PCR-amplifiable DNA molecules, which are those suitable for next-generation sequencing reactions. Therefore, real-time PCR is better suited for predicting the utility of a DNA sample for NGS than the conventional methods mentioned above, which often lack the power or overestimate the amount of amplifiable DNA present in compromised samples.
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