After sending a DNA sample for sequencing, the resulting sequence had N's in the beginning and end of the sequence. I know the N's mean that the computer can't tell what the base pair is, but why is this?
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2 Answers
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Typically Sanger sequencing will run into a few errors. Sometimes the traces will overlap as below in red and the computer will call N. If you truly wanted to figure out the correct basepair, you can look at the trace.
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2$\begingroup$ I'll add on to this answer. A Sanger sequence is really only good for (at best) 800-900 bp. After this time, there is simply too little DNA left to have a reliable signal that you can confidently say is a specific nucleotide. A good habit to get into is always examine your chromatograms and trace, and reject everything after you start getting your first run of N's. I don't usually worry about my first N at the end of the read however because it can be attributed just to error, and the following bases are still confidently called. $\endgroup$– user560Apr 16, 2012 at 7:03
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2$\begingroup$ This is not really an answer. In effect it just says “it happens” but not why. The wisdom of crowds seems an oxymoron in this context. $\endgroup$– DavidMay 26, 2020 at 19:01
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As you accurately stated, N bases in sequence data generally means the software is unable to identify the base. N bases may appear at the beginning of the sequence result for a number of reasons. One reason would be purification of the amplified product before electrophoresis. Salts in the sample or a poor purification could leave excess dyes in the sample and appear as "dye blobs." Another reason is the software may have started analysis too soon before accurate sequence begins. Typically, quality sequence data begins 30 bases from the primer. N bases at end of the sequence simply could be the end of sequence data as stated earlier. Other reasons include hairpin loops and poly base regions that cause early termination. The best way to determine the cause is to look at the trace data.
