if the amplimer that you are sequencing is very long then there may be no overlap between F and R sequences. Alternatively there may be too many Ns at the end of one sequence so it will not align.The quality of the sequence is very important when aligning sequences and it is always necessary to trim the ends off sequences where there are too many ambiguous/wrong bases to get good alignment  If you attach the .abi sequence file for the odd sequence and for another sequence that it should align/overlap with it would be helpful.

Dear Paul,

Thanks so much for your reply! I did trim the ends of each sequence but perhaps, as you mentioned, the quality is simply too low. I have attached the F and R sequences for context. If the quality is too low, is rerunning the PCR reaction a viable solution?

Best,

Wesley

hi wesley,

I will answer in more detail later but the simple answer is that these sequences are not good enough to read properly or to align.The signal strength is too low compared to the background for most of the sequence. There can be many reasons for this which I ,or any one else looking at these electropherograms will advise on.

best wishes

paul

Dear         Wesley   ,

Thank you for the .abi files.

The electropherogram shows a good signal strength ( >800 ) and a good signal to noise ratio up to 30 bases then a rapid deterioration after 30 bases.

This can happen with secondary structure of the template dna in which case adding 1m betaine to the sequencing reaction helps but as your other sequences work well I do not think this is the case.

Neither is there a homopolymer string which can cause this effect in your sequence.

Some possibilities assuming that you are using PCR product to sequence:

The sequencing primer binds to two (or more) sites on the template.

There are two (or more) templates present.

This was a PCR reaction, and you didn't remove the original primers.

This was a PCR reaction, and one primer generated *both* ends.

This was a PCR reaction, and there is more than one amplified species present.

You had a primer dimer which was not removed in template purification

Sometimes a pcr primer that is unique at high annealing temperature anneals at 2 positions at the lower annealing temperature of the sequencing reaction I do not think this is the case with you but check the sequence

If there are 2 similar templates in your pcr band then you can get this effect because after a few bases you are sequencing 2 different sequences. Check that your pcr band is the same size and clean as the others that work..

If your cleanup reaction /method did not remove all pcr primer then 2 sequences will be generated.

If one primer generated the band then you will be sequencing forward and reverse at the same time….I do not think this is the case with your sequences.

The concentration of the template or sequencing primer is wrong. If you have the wrong concentration of either template or primer it can lead to depletion of primer in the sequencing reaction so leading to too many short sequences and not enough long terminated sequences so a rapid drop off of signal strength. This seems likely with your sequence please check the concentrations of each.

If you have primer dimer present then you get a good signal strength sequencing the primer dimer then low signal strength of your template sequence. This looks possible. If you look at the .abi file you will see that the average c,a,g and T intensities are 507,538, 563 and 430. These are too low and should be in the 1000-2000 region for good sequencing.

This might suggest that removal of salt has been incomplete. Sequencing relies on electrokinetic loading of the dna in the sample. If there is any salt in the dna then the salt loads preferentially and only the short dna sequences load well. This may be the case with this sample. Sometimes all of the salt load and simply re loading the same sample on the sequencer gives you much better results as the first run has effectively desalted the sample.

My advice is check the quality and quantity of the template and primers. Possibly run a few more cycles in the sequencing reaction and make sure that the sequencing reaction is thoroughly desalted before loading on to the sequencer

Regards

Paul

Dear Paul,

I cannot thank you enough for your extraordinarily detailed and well thought out response! I will try what you have suggested and see if this works. 

In terms of primer concentration, what would you recommend? At the moment I am using 10 micromol of F and R for PCR. 

Thanks again!

Hi Wesley,

I had another look at the abi file and the current is steady during your run so I think that your desalting procedure prior to loading the sample is fine. generally you could increase the loading time of your samples if the signal is weak. Your sequence looks quite good but too weak around 600 bases so things are nearly right.

for a 25 ul pcr use .005-1 micro molar primer usually 0.2 umolar eg 0.5ul of a 10uM primer.

use 3.2uM concentration of sequencing primer ( 3.2pico moles of primer for abi3.1 sequencing kit.

use about 5-20ng of pcr product for sequencing 500-1000 bases or 200-500 bases of sequencing use about 3-10ng.

If your pcr usually works with the primer concentration that you are using then it is likely that this is fine and there is a problem with the dna that you are amplifying in this case. maybe some rna present so you have too little dna or possibly the dna has inhibitors in it.  If there is too little dna then use more inthe pcr but if you suspect inhibitors in the dna use LESS dna and it will amplify better. sometimes increased mg ions help with inhibitors. I would try a pcr with your dna template as before, also half that amount, also twice that amount with an extra 5 cycles of pcr and see if you get a better yield of product and maybe a sample with a little more mg ( if you use 1.5mM then try 2.0 mM abd see if it helps (usual amount of dna template)

good luck,

Paul

typo in the above answer .I meant 0.05-1 micro molar in the pcr mix not 0.005 as I posted

many thanks for this clarifications

Very nice explanation.

Purification of the PCR product prior to sequencing is quite important to avoid mismatching.

Regards