Troubleshooting, please help

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jfrose
jfrose's picture
Troubleshooting, please help

I have encountered a few sequencing problems recently. I sequenced the SAME PCR product (after exoSAP cleanup) in both the Forward direction and Reverse directions that are done in SEPARATE plates. Forward sequence in okay, but there is about 10 to 30 bases that are not sequenced well on the Reverse direction. It first become extremely slanted, and then it becomes "double peaks", for example, for location expected one T, it becomes two T, for locations expecting 3 A, it becomes 4 A.

Another weird thing is that the "bad chunks" seems to be around the same region across different samples. I did think about secondary structure, but we have sequenced the same region using the same method previously and this problem just comes up recently. For that, I also ruled out things like annealing temperature, primer design.

Another problem I encountered is premature stop. Recently, most forward reactions stop prematurely after 200 bases (where usually it goes up to 700 to even 900 bases).  It either becomes messy abruptly, or dies off gradually (first slanted peaks then beomce mixed).

Anyone has a clue about the cause of the problems?

Many thanks.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi ifrose,
The sequencing issues that you describe are ones that I have seen many times in the past. For some reason that I cannot completely explain, reverse reactions tend not to give as good quality sequence data as compared to forward reactions. This can be due to poly A tails (the multiple Ts and/or As that you describe) as well as other repeat sequences. One trick to get around this is to use two forward primers: one from the 5' end and another towards the end of your insert, but about 500 bp upstream of the end of the insert.
As for the sequencing run going for 200 bp and then crashing, that is typically an issue with too much template. The reaction generates tons of signal for the first 100 to 200 bp and the reagents get used up because of all the template. Typically if you reduce the amount of DNA you add to the reaction this goes away. 
As for your bad chunks, I am not sure what to say. I can't really think of anything other than high GC regions that would lead to compressions like these. Yet, from your description it does not sound like this is the case.

Rajiv-Genetics
Rajiv-Genetics's picture
Hi jfrose,

Hi jfrose,
For the abrupt stopping of your forward reactions, like Ivan mentioned it could be the template concentration. But since you mention it becomes messy with mixed peaks, There is a possibility of template contamination too. Since you do mention that it was working initially. Please do try using a freshly amplified template.Do you think there are primer dimers in your amplification.
Also the bad chunks that you observe on reverse sequencing is that in and arond the region where the forward reaction is dropping off?
 
Ivan, i have done some sequencing not an awful lot. But it is quite surprising that reverse sequences are not sequenced properly. I did observe that when sequencing my amplicons. Though i did get the sequence without a problem using both primers, the sequence obtained using the reverse primer didnt seem that great with good peaks in comparison to the forward reaction. I dont know if poly A has to play a role maybe it does but in my case the sequence was a part of a gene so no Poly A tail around the primer binding site. I had amplified the gene in smaller fragments to enable me for polymorphism/ mutation screening.
 

ryan_m
ryan_m's picture
In addition to the

In addition to the possibility of this being a polyA issue, it might also be what we refer to as a "hard stop".  There is information on this along with possible solutions here:
 
www.nucleics.com/DNA_sequencing_support/DNA-sequencing-hard-stops.html