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Addgene

Sequence Analysis of a Plasmid

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Introduction

Sequence verification of important plasmid features (such as the gene/insert, fusion proteins, point mutations, deletions, etc.) involves selecting one or more unique oligonucleotide primers that flank the regions of your plasmid that you wish to confirm. Addgene's plasmid information pages recommend 5’ and 3’ sequencing primers. These primers typically anneal to the backbone and can help you verify the ends of the insert. Longer genes may require internal primers that anneal to a specific region of the insert.

You can find Addgene's sequencing results by clicking on the "View Sequences" link on the Plasmid Information Pages. Addgene sequences the plasmid to verify tags, mutations and a portion of the insert, but we do not sequence the entire plasmid. Addgene strongly recommends that you sequence any portion of the plasmid that is important for your experiments.

There are a number of factors to consider when selecting sequencing primers, including:

  • Is the primer unique?

    Make sure the primer only anneals once within the entire construct.

  • Is the primer an appropriate distance from the target?

    The primer should be a minimum of 50 nucleotides and a maximum of 300 nucleotides from your target.

  • Are there commercially available primers that can be used for sequencing?

    There are many widely used, common primers that are often found within the plasmid backbone (See Addgene's Sequencing Primers for reference). Many sequencing cores have a list of common primers that can be requested for no additional charge.

  • What primers were used by Addgene during quality control?

    Addgene lists the primers used to obtain each result above the posted sequence in the "View Sequence" link.

  • Is your custom primer found within the Addgene sequencing results?

    If you require additional sequencing and need to design a custom primer, Addgene recommends using Addgene's sequencing results as a reference for primer design.

A good sequencing reaction will produce between 300-900 base pairs of useable sequence. You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks corresponding to one of the four nucleotide bases. This is an example of a trace file from a high-quality portion of a sequencing reaction:

A trace file that shows a sequencing reaction with easily distinguishable peaks that are discrete and above background.

Sequence near the beginning or end of a sequencing reaction is often unreliable. Although your sequencing results may indicate bases at specific locations, by looking at the trace file, you will see that these base calls are unreliable. This is an example of a trace file from a portion of a sequencing reaction with high background:

A trace file that shows a portion of a sequencing reaction with poorly distinguishable peaks that are misshapen and contain secondary peaks.

If you obtain a different sequence than expected and wish to contact Addgene about the accuracy of your plasmid, please email [email protected] and include:

  • The sequencing result
  • The original trace file (.ab1 file) provided by your sequencing company or core facility
  • The corresponding primer sequence used for each reaction

Tips and FAQs

My sequence doesn’t match Addgene’s sequencing result, what should I do?

Check your trace file first; the apparent mismatch/mutation may be the result of a mis-called peak in the trace file. If the mutation is not an artifact, please email [email protected] with the sequence, trace file, and primer used.

What program can I use to view my trace file?

There are many free programs available that can open .ab1 files, such as 4Peaks (Mac), SnapGene Viewer (Mac/PC), FinchTV (Mac/PC), Sequence Scanner (PC), and Chromas (PC).

How can I tell if a peak is mis-called?

Open the trace file and use the search feature in the program to locate the incorrect sequence. Look at the peaks in the area and make sure they are justifiable peaks. For instance, in the trace file below, you can see that just after base 70 there are multiple peaks in the same location. Looking at the trace file will give you more information than simply looking at the bases provided by your sequencing provider.

A trace file that shows a portion of a sequencing reaction with easily distinguishable peaks that are discrete and above background, except in one location where there are multiple peaks.

My sequence has “N”s in it- what does that mean?

“N” is the letter used to denote that the position could be any of the 4 bases. Check the trace file and see if you can manually call the correct base at the position. Sometimes an “N” is the result of an erroneous insertion by the sequencing reaction.