The presence of salt in your samples exacerbates this effect. This is normal and is a consequence of the capillary electrophoresis technology currently in use. Note that the 3730 tends to have low baseline noise early in the chromatogram, but it may increase modestly towards the end of the run. Don’t try to read baseline noise for usable data! Go to the Troubleshooting Guide for hints on what caused your lane to fail. If your signal is extremely low (anything below G=50, but on some sequencers even G=100) and your peaks are uninterpretable, then you should consider the lane to be simply blank – i.e. Signals between 50 and 100 SOMETIMES give reasonable data (and sometimes give poor data, as depicted in the above window), but you’ll definitely see baseline noise that would never trouble you on a bright sample.
On a 3730, this number should ideally be between 5 for best sensitivity and low noise (although this can vary significantly from instrument to instrument, or depending on the alignment procedures used to ‘tune’ the sequencer). In our lab, we usually just look at the first number, the ‘G’ signal, for simplicity. Here are some example signal strength numbers, reflecting arbitrary “relative fluorescence units”: Signal G:131 A:140 T:98 C:78 They are usually available in a separate pull-up window via your chromatogram viewing program, or if you’ve printed the chromatogram, they may be printed at the top. Check the ‘Signal Intensity’ numbers for your chromatogram. Noise like the above most commonly arises when the sample itself is too dim. Note the multicolored peaks at 271, 273 and 279, the oddly-spaced interstitial peaks near 291 and 301, and it is impossible to determine the real nucleotide is at 310. Now we get to an example that has a bit too much noise. The next example has a little baseline noise, but the ‘real’ peaks are still easy to call, so there’s no problem with this sample: Note the evenly-spaced peaks and the lack of baseline ‘noise’ (see further down for examples of higher baseline noise): Here’s an example of an excellent sequence. If your results do not fit this description, consult our Troubleshooting pages. “Noise” (baseline) peaks may be present, but with good template and primer, they will be quite minimal. Peak heights may vary 3-fold, which is normal. Get a General Sense of How Clean the Sequence Is How clear are the nucleotide peaks, in general? You should see evenly-spaced peaks, each with only one color. It is not necessary to read each and every base.ġ.
With a little practice, you can scan a chromatogram in less than a minute and spot problems. Occasionally, information in this document will refer to older sequencers for historical purposes, and we will specifically state when this is the case. The current version of this document reflect our current generation of DNA sequencers, the ABI Model 3730XL. truncate the sequence when errors become too frequent.It is the responsibility of the Core clients to: Links are also given for more complete troubleshooting of problems.
This document explains how to examine the normal DNA sequencing chromatogram, describing common issues and how to interpret them. Other errors can crop up in the middle, invalidating individual base calls or entire swaths of data. Predictable errors occur near the beginning and again at the end of any sequencing run. That computer program, however, does make mistakes and you need to manually double-check the interpretation of the primary data. Truncate the sequence where problems become too frequentĪutomated DNA Sequencers generate a four-color chromatogram showing the results of the sequencing run, as well as a computer program’s best guess at interpreting that data – a text file of sequence data.Get a general sense of how clean the sequence is.