Interpreting the Peaks: Reading DNA Chromatogram

If the sequencing machine detects a flash of fluorescent light as a DNA fragment passes the sensor, then it records that event as a peak. If each peak corresponds to a specific dye-labeled nucleotide, then the peak represents a base like A, T, C, or G.

If each of the four deoxynucleotides is tagged with a different colored fluorescent dye, then the sensor can distinguish them by wavelength. If the color is unique to the base, then color identifies the nucleotide.

If the industry standard for fluorescent labeling assigns green to a specific base, then Adenine is the base represented by that color on the plot.

If two or more peaks overlap or the signal is too weak for the software to choose a single base, then it assigns a placeholder. If "N" is the universal symbol for "unknown," then it indicates an identification failure.

If the standard dye set is used, then A is green, T is red, C is blue, and G is black or yellow; therefore, matching Red with Guanine is an incorrect interpretation.

If the fluorescent signal is strong and distinct from the background noise, then the peak will appear tall and narrow. If the peak is well-defined, then the computer can accurately identify the base.

If the bases are plotted from left to right as they pass the detector, then the horizontal distance corresponds to their numerical order in the sequence.

If an organism has two different alleles for a gene, then one chromosome may have an 'A' while the other has a 'G'. If both are sequenced simultaneously, then two overlapping peaks will appear at that single position.

If the reaction contains impurities or incorrect starting materials, then the fluorescent signals will clash or drown each other out. This creates random small peaks known as baseline noise.

If DNA polymerase builds the new strand by adding nucleotides to the 3' end, then the smallest fragments (the beginning) are the 5' end. Since small things move fastest in electrophoresis, the 5' end is read first.

If more DNA fragments of a specific length are present and glowing, then the sensor will pick up a stronger light signal. If the signal is stronger, then the peak height on the vertical axis increases.

If excess fluorescent dye molecules clump together and pass the sensor without being attached to DNA, then they create a massive, non-specific signal. If this signal is wide and bright, then it obscures the underlying data.

If a computer evaluates the peak shape and resolution, then it assigns a score to tell the scientist how much to trust that base. If the score is high (like Q30), then the error probability is very low.

If the graph represents the chronological arrival of DNA fragments at the detector, and if time moves forward on the x-axis, then the sequence must be read from left to right.

If the DNA was separated by an electric current (electrophoresis) and the result is a visual graph (gram), then the scientific term for this specific image is an electropherogram.

If larger DNA fragments move more slowly and diffuse more during electrophoresis, then they hit the sensor with less precision. If they are less precise, then the resulting peaks will be spread out and harder to distinguish.

If a base is deleted or inserted in one allele, then the two strands will be "out of sync" for the rest of the run. If they are out of sync, then the machine will detect two different bases at every single position thereafter.

If a run is clean, then the area between the peaks and the baseline should be empty. If minor random signals are detected, then they appear as tiny "grass-like" bumps along the bottom of the graph.

If the amount of light exceeds the sensor's ability to measure it, then the top of the peak is flattened. If the sensor is overwhelmed, then the signal is described as being saturated.

If the goal is to determine the sequence, then you must find the dominant signal at each point. If you also check the Phred score, then you ensure that the "call" you made is mathematically reliable.