Can you attach one of the original .abi sequence files from the sequencing company please?. They often contain useful information. How much dna are you sending for sequencing and how much primer? What is the annealing temperature of the primer? What method of pcr product cleanup are you using?

Primer amount, DNA amount and DNA concentrations as follows,

matK kim M13 (FP and RP) - 0.6 ul

Di_3 - 8ul of 55.8ng/ul DNA

Di_5 - 8ul of 52.3ng/ul DNA

Di_7 - 8 ul of 126.5ng/ul DNA

Trnh-psba (FP and RP) - 0.6 ul

Da_1 -0.5ul of 1367.0ng/ul DNA

Da_2 - 2ul of 676.3ng/ul DNA

De_1 - 2ul of 672.9ng/ul DNA

For matK kim M13 I extracted DNA from roots of the Dioscorea.spp and for Trnh-psba I extracted DNA from the lraves of the Dioscorea.spp

Annealing temperatures

matK kim M13- 55C

Trnh-psba- 50C

30ul of primer was sent

I didn't used any cleanup methods for PCR products.

I have attached original abi files with this also

Thank you for the files Samitha Disanayaka .I will get back to you about these later to day but one more thing will help please. Do these samples have visible primer dimer ( a diffuse weak band at below 100bp) in the checker gel of the pcr product that you sent for sequencing?

Thank you for your kind attention @Paul Rutland. Yes, there were visible primer dimers of the pcr products.

Thank you Samitha Disanayaka . I will look at your sequences in detail later but something to think about. P-d is very short in length so does not trap much EtBr so looks quite faint and it also spread out due to being small and thermal diffusion. This means that there is a lot of it even though it looks faint and worse still it has perfect homology with your primers. So in a sequencing reaction the p-d will bind your sequencing primer very well and the amount of p-d which melts easily and sequences short and well will remove most of your sequencing primer before the real template has a chance to generate a strong signal. At least one of your sequences has a perfectly good sequence up to 250 bases but very weak after the first few bases suggesting that in at least one of your samples p-d is responsible for poor sequencing. I will look more closely at your samples and get back in a few hours

Thank you Paul Rutland for your suggestion. Actually there were very clear bands of p-d in gel image as clear as the pcr product except for Di_3 sample.I used SYBR safe as the stainer.

Hi Samitha,

Some thoughts on your sequences.

DA1TRNHPSBAfp

Starts with strong (>1000) well spaced base peaks dropping slowly from base 105 to about 330 bases with a signal/noise of 200. Quite good sequencing but this kit is capable of 900 bases of strong intensity signal. This is usually cause by insufficient primer or template. If this sample has primer dimer ( PD) then this will measure as DSdna so less template will go into the sequencing mix and the signal will die early and the remaining template will give a weak but readable signal

DA2TRNHPSBAFP

The signal strength is only 28 and s/n ratio is 4 so no readable sequence at all.The reaction has failed completely so possibly primer and template do not match

DI5MATKKIMM13FP

Clean strong base peaks up to base 53 ( strength 1600)then signal drops to zero.

This can happen with some secondary structure problems in the template dna but can also happen if you run out of primer. In this case I suggest that the sequencing primer has been used up sequencing PD

DI7MATKKIMM13FP

Good clean strong ( 3700) sequence up to 46 bases then dropping to baseline intensity. There is no huge unincorporated dye peak so the sequencing reaction is working but clearly no on the template dna so I think that we are sequencing a strong PD

De1TRNHPSBAFP

Very large dye peak ( mixed bases) at start of sequence (>4500) then no peaks. The sequencing reaction failed completely. Either no primer, no template or the wrong primer used in sequencing.

D13MATKKIMM13FP2

160 strong bases all Ns. We have 2 primers annealing in different places and producing 2 sequences. As it is unlikely that you supplied 2 templates so something is acting as a second primer or one primer is annealing in 2 places on the template dna ( unlikely), The sequence stops much too early. I do not know the cause for this one but if you had not told me that there was no PD I would say the pd has dissociated and caused F and R primers to anneal to the template and has used up the sequencing mix too early.

RecA C92f

Huge unincorporated dye peaks (17000 and 4500) at ends of sequence with low intensity clean sequence in between. I think that you have light PD which has sequences and used much of the sequencing primer and the rest has produced a weak but accurate sequence that has not been basecalled. The reason for not calling the bases is that the unincorporated base peaks are so high that the base calls are below the set cut off value of 5% of the highest peaks so they are ignored.

RECAC92F

Huge (>12000) unincorporated dye peak at start dropping very rapidly after 35 bases. Dye peaks at 85 and 120 bases indicate that the sequencing has failed. I think the primer was all used on the PD sequence and the unincorporate dbases are from the unused sequencing mix

Overall I feel that your problems are caused by PD using up all the sequencing primer not leaving enough to get a good signal from the template dna. Additionally pd is double stranded so will measure as DS dna when calculating the amount of dna to sequence so you are actually sequencing too little dna.

You may need to clean up your pcr ( use less primer for a start) or gel separate the template and clean it up before sequencing.