Hi Avin,

As the other commenters have mentioned, this is perfectly normal.  You will generally get about 500-800 nt of reliable sequence read from the dye-terminator sequencing.  You won't get the primer sequence, only the sequence 3' of it after a few nucleotides (the resolution is generally a little iffy immediately after the primer), and the resolution of individual peaks/bands also makes it hard to "call" bases with accuracy after 500-800 nts.  You could solve this problem by (1) either using nested internal primers, or (2) subcloning your PCR-ed fragment into a vector and then using sequencing primers from the vector itself (most vectors have some common primer sequences flanking the multiple cloning site).  

Best of luck.

Hi Avin,

When you are sequencing, often there are errors or truncations right beside the primer binding site.  This is why when we are sequencing, we design primers that are at least 50 bp outside of the region of interest.  Also, theoretically you should not actually see the primer sequence (assuming you are using dye-terminator sequencing) in the chromatogram.

How far outside of the gene of interest do your primers bind?

Good luck

It's usual thing for long reads at least for sanger sequencing. How long the PCR product you sequenced and what kind of sequencing you use (Sanger, Illumina or smth. else)? As for sanger sequencing it will depends of capillare array lenth and ferment activity (at least you will obtain 600-800 bp. of good read). If your sequences more long you may try to  use of internal primers for obtaining good quality reads for all lenth (easiest and chipest way) or change cappilare assay in sequencer (for example from 36 cm to 80 cm), but it is much more expensive . The fact that you can't find forward primer also not surprizing because in sanger technique you have start with not good quality in most cases. But if you use another techniques (i.e. Illumila or others) there another features and factors may explain this fact. If you provide more information you can get more precise answer.

Thanks for the prompt replies,

If I'm not mistaken, it should be dye-terminator sequencing. The primers are actually binding right at the start and end of the gene, roughly around 10 bases away.

Dear Avin

These problems are common, I have faced a similar kind of problem sequencing a 1208 bp fragment of COI gene and I end up obtaining 900 bp sequence when done on ABI 3730 Automated DNA sequencer. Try to design internal primers and cut down your fragment length to

This is absolutely normal (I assume your are using your PCR primers for sequencing). This phenomenon is basically due to the (low) resolution of the sequencing gel for short molecules. Besides, the sanger method doesn't generate the sequence of the primer used for sequencing but for the template DNA, starting from the first nucleotide after the 3' end of the primer. Improving template purity is always a good thing for longer sequences and sharper, cleaner chromatogram peaks.

Hi Avin,

As the other commenters have mentioned, this is perfectly normal.  You will generally get about 500-800 nt of reliable sequence read from the dye-terminator sequencing.  You won't get the primer sequence, only the sequence 3' of it after a few nucleotides (the resolution is generally a little iffy immediately after the primer), and the resolution of individual peaks/bands also makes it hard to "call" bases with accuracy after 500-800 nts.  You could solve this problem by (1) either using nested internal primers, or (2) subcloning your PCR-ed fragment into a vector and then using sequencing primers from the vector itself (most vectors have some common primer sequences flanking the multiple cloning site).  

Best of luck.

Hi 

It is NOT normal to get shorter sequences.

You should go for 2-way sequencing in order to obtain full length if your target is >800bps.

In 2-way chromatograms you should be able to see the respective primers.

cheers

Hi 

You can always try to past your sequence into for example pGEM vector and transform E. coli. After isolation of plasmid with Your sequence from E. coli for the sequencing use primers SP6 and T7 then u will avoid shorter sequences. Remember also that Taq polimerase insert errors into sequence so for the sequencing the best is Pfu polymerase. 

Good luck

It seems that you are using PCR product for sequencing. Have you tried to overlap the forward and reverse sequencing results? If a few base pairs from the froward primer are missing, you should be able to get that sequence at the end of your reverse primer sequence. Usually we don't look for primers in the sequencing. It also depends upon the length of the gene to be sequenced. If it is very long, it is normal to get truncated sequencing results. I usually design some internal primers to sequence a long gene.

It is not normal to see truncations in your PCR fragment.  This may be occurring during the PCR amplification and may suggest some repeats in the primer.  It is normal for you to get poor or unreadable sequence within 1-30 bp of the end of the primers.  There is not enough dye incorporation at that point to get reliable sequence data.

Also, please make sure you remove the PCR primers before sequencing.

First of all , you have to clone your PCR product on a cloning vector . For example pGEM-T easy vector and transform the recombinat vector to E.coli. For sequecning, i prefer to use the universal primers of teh vector NOT the primer used during PCR amplifciation. Thus any nise in the begining or at the end of the sequence will appear in the universal primers already existed on the vector. In case of pGEM-T vector , the universal primers are sp6 and T7. If the problem still exists in this case. You have to do further step. You already got the sequene. So based on this sequence , you have to design internal primers to the front and to the back. I mean Internal forward primer (IFP) and Internal Reverse Primer (IRP). For example, design an IFP and position of Nucleotide 700, then upon sequencing it will go to a considerable distance giving you the end of the gene without any problems. Design another IRP at position for example, Nucleotide 300 then upon sequecning, sure it will go to the back and cover the whole sequecne from the begining. Hopefully i could tell a right answer.

For sequencing of short or long amplicons, I suggest cloning of the gene in any T-vector (pGEM or Topo-TA), amplifying the plasmid, and then using the M13 sequencing primers for sequencing. This not only improves sequencing efficiency, but also renders in clean chromatograms. However, plasmids should be PEG-purified to achieve clean results.

Thank you all for such helpful advice, especially the bit about using internal primers. 

Cloning is not incorrect, but you really do not need to clone the PCR fragments in order to get good sequencing results.   Just purify the PCR products over a Qiagen mini column to get rid of the PCR primers and then sequence the fragment with two internal primers that read from the middle to the ends of each strand.  With enough overlap in the middle you need not worry about the first 30 bp after the 3' end of th sequencing primers. 

Yes, cloning would solve the problem but is probably surplus to requirements as mentioned

Loss of sequence immediately adjacent to the priming site is not uncommon

Apart from column purification as mentioned to recover these primer adjecent dye labelled sequencing  products,  if you are in the habit of ethanol ppt, use of MgCl2 instead of Nacl to ppt the dye labelled fragments has allowed me in the past to read all the way back to the priming site. It is actually an acknowledged method for precipitating very short DNA fragments but like alot of these older manual methods in this age of convenience columns tends to get forgotten. Let me know if you require details

One more thing has occured to me in terms of losing signal at the 3' end of your product; Is your raw data very top heavy. If so it is not usually the quality of DNA templete that is at issue. Rather the quantity. Massively over loading your dye labelled DNA sequencing reaction leads to sequestration of raw materials during intial synthesis of 5' products and depletes materials for subsequent synthesis of longer more 3' products leading to over represenation of the former and under represenation of the latter. How do you quantitate your DNA template. How much do you add to your sequencing reaction ?

Hi Alvin

 It is common in dye-terminator sequencing to miss the primer sequence. you will also miss ~ 50 nucleotides on either side of the chromatogram because of the signal pickup issue of chromatogram. it also shows mismatch at those regions. so you should design your primers leaving ~ 50-100 nucleotides before and after to your region of interest. the preferred PCR product size for sanger sequencing is 800 bp including primers and flanking regions.

Have you checked your PCR product size on gel before sending it for sequencing. If it is perfect and your sequence shows any drop off or mid sequence stop,then it is problem with the sequencing. you can ask for repeating the sequencing,which solves the problem most of the times.

good luck

What you are getting is normal. However, it is better. you still check your system ( I think there will be no difference). 

getting less sequence length could be due to high concentration of your product (so big dye terminator is exhausted early). so measure the concentration of the product before going for sequencing and add the product accordingly. choosing a constant quantity of PCR product (eg 2microliter of product) in the sequencing reaction may lead to this problem.

what is your product size. if product size is more than 600bp then you might not get your desired product with just forward and reverse primers. in those cases add internal primers.

regarding not getting the primer sequence is normal (as the chance of addition of ddntp at beginning of cycle is less (dntp and ddntp are added at 10:1 concentration).

you have also stated that you are getting many substitutions. for this you have to study the chromatogram and have to choose the correct base. when ddntp concentration decreases then the result you get is not perfect. therefore we donot get correct result in first 20- 50 bases of the sequencing cycle and then in the last part of the sequence. you have to see the size and clarity of the chromatogram and decide.