How to Read and Interpret Electropherograms

An electropherogram is a picture of a race. Molecules are pulled through a hair-thin tube, and each peak marks a group that crossed the finish line together. Reading one means asking three things of every peak: where it sits, how big it is, and whether it is real. The same kind of graph is used for DNA sequencing, forensic DNA profiles, RNA and DNA quality checks, and blood protein tests, and each one asks something different of the peaks.

What the Axes and Peaks Show

The sample travels through a narrow tube between 20 and 200 micrometres wide, under high voltage.1Capillary Electrophoresis Handbook (SCIEX). A Guide to Capillary Electrophoresis and Modern Techniques Charge and size make molecules move at different speeds, so they separate along the way.2Excedr. Capillary Electrophoresis Guide: Modes, Controls & A detector near the end of the tube records each group as it passes.3Wikipedia. Capillary electrophoresis The graph plots that detector signal against time, and separated compounds show up as peaks at different times.4Wikipedia. Capillary electrophoresis

Later peaks are usually larger or slower molecules, so a peak’s position, read against reference peaks, helps tell you what it is. Its height or area tells you how much is there. Area can mislead, though, because it depends on where the software decides a peak starts and ends.5Bio-Rad Laboratories. Development and Validation of RQI: An RNA Quality Indicator for the Experion Automated Electrophoresis System Many DNA tests also label molecules with different colored dyes, and software separates those colors.6PubMed. An estimate of the crosstalk matrix in four-dye fluorescence-based DNA sequencing

With an unfamiliar trace, start by finding the axes and checking whether the horizontal one shows time, base pairs or nucleotide position. Next, locate the reference or control peaks before looking at your sample’s peaks. Then compare peak heights to the baseline noise to judge which peaks are clearly real. Last, note color: in multi-dye runs each color is a separate channel.

Reference Peaks That Anchor Every Reading

In DNA fragment analysis, a size standard, a set of fragments of known size, runs in every tube in every run.7Forensic Biotechnology. How to Read and Interpret Electropherograms Software uses those ruler peaks to convert travel time into size in base pairs.8Forensic Biotechnology. How to Read and Interpret Electropherograms An allelic ladder, a mix of common DNA variants, lets the software match sample peaks to named versions.9Forensic Biotechnology. How to Read and Interpret Electropherograms Forensic analysts check the ruler peaks, the ladder and the controls before trusting any sample peak.10National Institute of Justice. STR Data Analysis and Interpretation for Forensic Analysts | Overview

Every expected ruler peak should be present and labeled. A missing or misplaced ruler peak makes all sizes in that run suspect. Controls should show exactly the expected result, with no extra peaks in blanks.

Anchoring can fail in other kinds of tests too. Hemoglobin tests use the normal HbA and HbA2 peaks as anchors to label every other zone.11PubMed. Capillary electrophoresis mis-anchoring in a case of Hb Hope with HbE If HbA is missing, or a variant moves like it, the software can mislabel the whole trace.12PubMed. Capillary electrophoresis mis-anchoring in a case of Hb Hope with HbE In one reported case, Hb Hope and HbE were mislabeled as HbA and HbA2.13PubMed. Capillary electrophoresis mis-anchoring in a case of Hb Hope with HbE

A mislabeled anchor is hard to see because the trace itself looks normal while the labels are shifted. The clues are results that clash with the patient’s history or with other tests. A recheck by a different method usually settles it.

Reading a Sanger Sequencing Trace

Each of the four DNA letters carries its own dye color, and software reads the color of each peak to call the letter.14ELECTROPHORESIS. Alternative base-calling algorithm for DNA sequencing based on four-label multicolor detection Wherever the called letters look surprising, look at the trace itself.15OpenAlex API / Journal of Biomedical Science and Engineering. Novel algorithms for accurate DNA base-calling

At each position you want one clean peak in one color. A dye blob is a multicolor spike from excess dye, usually near the start of the read.16PubMed Central. Mastering DNA chromatogram analysis in Sanger sequencing for reliable clinical analysis Two different-colored peaks at one position can mean the sample carries two versions of that letter, one on each copy of the gene.17PubMed Central. Mastering DNA chromatogram analysis in Sanger sequencing for reliable clinical analysis In organisms with a single copy of each gene, double peaks can signal a mixed infection.18PubMed Central. Mastering DNA chromatogram analysis in Sanger sequencing for reliable clinical analysis

Some stretches of a read deserve more trust than others. The first few dozen positions are often messy and blob-prone. The middle usually has the cleanest, evenly spaced peaks. Toward the end, peaks broaden and shrink, and calls become less reliable. Sequencing both strands helps confirm uncertain positions.

A true double peak has two peaks of reasonable height at exactly the same position. Noise produces small, scattered peaks under many positions, not just one. A real variant also shows up on the reverse-strand read.

Reading DNA Library and RNA Quality Reports

A sequencing library should show these features:

Small peaks matter because short fragments and leftover adapters get sequenced preferentially and waste space, which lowers the yield of usable data.23Yeasen – Leading Innovation in Molecular Enzymes and Reagents. Rescuing Your Failed Libraries: A Guide to Ugly Electropherograms and What They Mean A library may be rejected if short fragments exceed 3% of the total peak.24Yeasen – Leading Innovation in Molecular Enzymes and Reagents. Rescuing Your Failed Libraries: A Guide to Ugly Electropherograms and What They Mean Degraded starting material is a common cause of extra short fragments.25Yeasen – Leading Innovation in Molecular Enzymes and Reagents. Rescuing Your Failed Libraries: A Guide to Ugly Electropherograms and What They Mean

A failed library has recognizable shapes. A sharp peak near 120 to 130 base pairs often means adapter dimers. A broad hump far above the target suggests over-amplification or clumping. A ragged multi-peak curve points to uneven fragmentation.

For RNA, intact samples show two distinct ribosomal peaks, labeled 28S and 18S.26Bio-Rad Laboratories. Development and Validation of RQI: An RNA Quality Indicator for the Experion Automated Electrophoresis System A 28S to 18S ratio near 2 suggests intact RNA, but the ratio is unreliable because it depends on where the peaks are cut.27Bio-Rad Laboratories. Development and Validation of RQI: An RNA Quality Indicator for the Experion Automated Electrophoresis System Automated scores such as the RIN, an RNA integrity score from 1 to 10 in your report, are preferred.28PubMed Central. The RIN: an RNA integrity number for assigning integrity values to RNA measurements

Degraded RNA looks different: the two ribosomal peaks shrink and lose their sharp shape, and a smear of signal grows toward the short-fragment end. The RNA integrity score drops as that smear increases.

Reading a Forensic DNA Profile

Labs set a detection cutoff below which a peak is not called.29National Institute of Standards and Technology. Standard for Determining Analytical and Stochastic Thresholds for Application to Forensic DNA Casework Using Electrophoresis Platforms A second, higher threshold flags when a true allele may have been missed.30National Institute of Standards and Technology. Standard for Determining Analytical and Stochastic Thresholds for Application to Forensic DNA Casework Using Electrophoresis Platforms

The cutoff is a trade-off, not a quality grade. Set higher, fewer noise peaks get called as alleles, but small true peaks from a minor contributor or a degraded sample can fall below it and go uncalled. Set lower, it captures more of those weak true peaks, but it also lets in stutter peaks, spikes and background noise that an analyst must sort out. A reported profile is only as complete as the cutoff allows, and a missing peak does not prove the person’s DNA was absent; it may simply have been too small to call.

In a single-source profile, each location shows one peak if both parents passed on the same version, or two similar-height peaks if not. Very uneven paired peaks or more than two clear peaks at several locations suggest a mixture.

In samples with very little DNA, alleles can drop out, and stray alleles can drop in.31PubMed. Production of high-fidelity electropherograms results in improved and consistent DNA interpretation: Standardizing the forensic validation process32PubMed. Interpreting low template DNA profiles Counting alleles can underestimate the number of people in a weak mixture by more than two.33PubMed. Determining the number of contributors to DNA mixtures in the low-template regime: Exploring the impacts of sampling and detection effects A profile that looks like two people may come from four or more.

Spotting Fake Peaks and Baseline Problems

Color bleed, also called pull-up, happens when software fails to separate dye colors.34National Institute of Justice. STR Data Analysis and Interpretation for Forensic Analysts Overloaded, very tall peaks also bleed into other colors.35National Institute of Justice. STR Data Analysis and Interpretation for Forensic Analysts A blue peak can then create a false green or yellow peak at the same spot.36National Institute of Justice. STR Data Analysis and Interpretation for Forensic Analysts So check every small peak for a large peak of another color directly above or below it.37National Institute of Justice. STR Data Analysis and Interpretation for Forensic Analysts

Stutter peaks are small peaks just next to a true STR peak, made during copying.38Forensic Science International: Reports. Developmental validation of FaSTRâ„¢ DNA: Software for the analysis of forensic DNA profiles Dye blobs and spikes are other common fakes in DNA profiles.39Forensic Science International: Reports. Developmental validation of FaSTRâ„¢ DNA: Software for the analysis of forensic DNA profiles40PubMed. Interpreting low template DNA profiles

Each fake has a signature. Color bleed lines up exactly with a big peak in another color. Stutter usually sits one repeat unit shorter than the real peak and is much smaller. A dye blob is broad and fuzzy rather than sharp. A spike is a very narrow peak appearing in all colors at once.

Baseline problems are a separate trouble. In some protein tests the baseline drifts because the buffer heats up from the current.41CASSS. Troubleshooting CE-SDS: baseline disturbances, peak area repeatability and the presence of ghost peaks Software can remove noise and correct drift mathematically.42PubMed. Signal denoising and baseline correction by discrete wavelet transform for microchip capillary electrophoresis

Decide whether a peak is real before comparing it to a reference sample. Ask a second person to review borderline peaks.

Blood Protein and Hemoglobin Electropherograms

In a protein trace, a sharp extra band, called an M-spike on reports, signals a large amount of one identical antibody made by a single group of cells.43PubMed. Challenges of measuring monoclonal proteins in serum Its size helps measure how much abnormal protein is present.44PubMed. Challenges of measuring monoclonal proteins in serum A follow-up test called immunofixation identifies its type.45Elsevier. Monoclonal gammopathy detection and current technologies

Hemoglobin traces show peaks for normal and variant types.46PubMed. Capillary electrophoresis mis-anchoring in a case of Hb Hope with HbE Some variants appear in the same place, such as HbC and HbE, or HbS and HbD.47PubMed. Quantitative and qualitative analysis of haemoglobin variants using capillary zone electrophoresis A second test under different conditions is needed to tell them apart.48PubMed. Quantitative and qualitative analysis of haemoglobin variants using capillary zone electrophoresis

Useful questions for your doctor: whether a spike was found and how large it is, whether the hemoglobin variant was confirmed by a second method, and whether the result fits your blood count and family history.

Can Software Read It for You

One forensic program correctly labeled 99.3% of stutter peaks in single-person profiles.49Forensic Science International: Reports. Developmental validation of FaSTRâ„¢ DNA: Software for the analysis of forensic DNA profiles It found true allele peaks in reference profiles with 99.7% accuracy.50PubMed. Validation of a neural network approach for STR typing to replace human reading A neural network program performed as well as analysts on case data.51PubMed. Making AI accessible for forensic DNA profile analysis

Software still struggles with low-DNA and mixed samples near the cutoff, and with unusual variants that don’t match the reference peaks. It can also miss its own mistake when it has labeled an anchor peak wrongly.

Treat automatic calls as a first draft. Review every flagged or borderline peak by eye, and keep the raw trace, not only the summary table.