- 4 of them have a Homopolymeric Region followed by Noisy Multiple Peaks. An example image for one of the samples of this case is attached with both the sequences of its Forward and Reverse Primers.
- While, the other 4 have a Homopolymeric Region followed by Lost Signal (Flatline/No Sequence).
Do you believe these 8 cases represent:
- Homopolymeric Region, thus i need to redesign Primers? OR
- Heterozygous or STRs case?
the most likely reason is that the homopolymer is polymorphic so that in both forward and reverse you will get 2 sequences after the end of the shorter poly base so every base position will have 2 bases of lower intensity than the single bases. you can check this by listing both bases at every position and you will see the expected sequence and also the sequence with an inserted extra base. It is a good and expected result. The 2 sequences can be checked by cloning and sequencing clones but you should see both sequences quite clearly by carefully analysing theses electropherograms
It is not clear to me whether the heterogeneity of the homopolymer is genuine or due to some slippage of the DNA polymerase used for sequencing. This could be tested if you follow Paul's advice and reclone the DNA used as a template (of course, this does not apply if your DNA is already derived from a clone). If the chromatograms of cloned are still ambiguous, then it is likely that the sequence heterogeneity is a sequencing artefact. If not, then the heterogeneity must lie in the original template.
Do these sequences derive from a PCR amplicon? Possibly, you are amplifying multiple copies of a repeated element that is somewhat polymorphic. Cloning and resequencing several clones would indeed help. Looks like a highly repetitive sequence, with a lot of homopolymeric stretches
I overlooked the part of the question about the flatlining after a long homozygous homopolymer in some samples. Can you post a .abi sequence file of one of these please? It is possibly reagent depletion and soluble by using more sequencing mix or some phenomenom like G quadruplex causing a structural termination of the sequence reaction so adding DMSO to the reaction may help but it would be interesting to see the raw data
good remarks by Paul and Pierre. Concerning the flatlining after the homopolymer cluster, it is very noticeable on the first chromatogram of Sarah (position 180, start of the reaction?) less on the second chromatogram (position 400, end of the reaction?)
Thank you professors.
@Dr. Muller, yes these are sequences are for PCR Products.
@Dr. Rutland, here is an image for one of the samples with a flattened/No Signal sequence after a Homopolymeric Region.
In your first comment, you said: "..can check this by listing both bases at every position and will see the expected sequence and also the sequence with an inserted extra base." How can i do this step, Do you mean sequence alignment? I already made a BLAST and other alignments for the Forward Primer-based Sequence and the Reverse Primer-based Sequence with the revised Cambridge Reference Sequence (rCRS). But weirdly, i found no fitting between the 3 sequences despite that the Homopolymeric Region is found, -as seen in the first image i attached with the question upwards-, with the same number of nucleotides and with complementary base pairs, Cytosines in the Forward Primer-based Sequence, while there are Guanines the Reverse Primer-based Sequence!
Dear Sarah,
Thank you for the image but the original .abi sequencing file does give much more information than just a picture.
What I mean by reading both bases is to read the sequence by eye and making a note of all of the bases ( 2 at every position after the poly G) then subtract the known sequence and what is left is the other sequence in this amplimer which will tell you the nature of the insertion/deletion in your sequence.
If I could give you an example of what I mean:
You have a sequence which should be GGGGGGGCATCATCATCAT
What you sequence is GGGGGGG(GC)(AC)(TA)(CT)(AC)(AT)(TC)(CA)(AT) where bracketed bases are both at the same position in the sequence.
You subtract the known sequence CATCATCATCAT from the bracketed bases
This leaves GCATCATCAT . This tells you that one G has been inserted into the polyG
It works just the same with small deletions.
Concerning sequence blasting the databases at NCBI are very good
https://blast.ncbi.nlm.nih.gov/Blast.cgi
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