1/ Do you get rid of the primers used in your PCR reactions prior to sequencing? (by e. g. gel-purification). If not, then the presence of two primers in the sequencing reaction will result in messy sequencing.

2/ I would recommend using a control sequencing reaction to be sure that all the components are OK.

3/ Your primers may find near target sequences in these bacteria. I would recommend designing new primers.

Are your no template controls clean ( no amplification) and is your pcr ban the correct size.

Can you attach a sequencing .abi file of a poor sequence please?

Is the pcr band a clean single band on agarose gel

Are you using the same primers for sequencing as you used for the amplification?

Are the pcr primers optimised...if the annealing temperature is too low then other sequences will amplify and the sequencing will be messy....if the pcr primers will work at a higher temperature then tis may be worth trying

Do you have any small band amplification...if you have primer dimer and if your primer clean up does not remove primer dimer then the sequencing will be poor...can we see a picture of the agarose gel of a poorly sequencing amplimer please?

Hi Nikita

it sounds like wrong purification of the PCR product, efficiency/specificity of your primers and contaminations by other species.

fred

Libor Krásný My samples were sent to a sequencing facility which did the Post PCR clean up and sequencing.@Libor Krasny

It would still be useful to see the original sequencing file which have much more information than just the electropherogram and it would also be relevant to know if your no template control has no amplified band in it to be sure that there is no pcr contamination present

In some instances my negative controls contained a faint band, however the bands for the samples were the correct size and single bands.

i am also using the same primers for sequencing as i am for PCR.

Attached is an example of bands in wells 1,3,6 and 16 all of which did not sequence but failed. 16 produced a messy short sequence seen in the attachment.

@Paul Rutland

Thank you for the files Nikita. Unfortunately I could not open the .scn file which is a shame because it is probably important concerning primer dimer (PD) but the .abi worked fine. Your signal intensities were about 32-124 ( 500plus is normal) with normal start scan time (no salt contamination) and background 8 ( normal) thus signal/noise ratio of 4-15 which is poor. The base spacing is 16 which for a 3730xl sequencer is very poor and the sequence stops at scan 2420 ( expected 18000). This is indicative of multiple (2 or more ) sequences being present and if the signal strength was higher I would say there are 2 sequences and the solution is to either clone and sequence or raise the annealing temperature to eliminate one sequence or redesign the primers.

The very short termination combined with lack of any good sequence suggests that the second sequence comes from primer dimer if pcr amplification was poor and primer dimer was produced in these samples. PD is short so traps very little ETBR so stains weakly but if you can see it at all then you have a lot of it. As it has the primer sequence incorporated it uses up all of the sequencing reagents very quickly so leaves very little for a good signal from your full length sample sequence. If the failed samples had poor amplification due to pcr inhibitors present in the dna then PD would form easier. If this is the case cutting the pcr band from a gel and purifying would help to get a clean sequence. Not a definitive answer by any means but I hope it helps

best wishes Paul

agree with@ Libor Krásný

regards