I do not think that this is a het because at 260 there is a run of 6Cs. If the sample was a het with 6 bases missing earlier in the sequence then these 6Cs would be duplicated 6 bases downstream and produce a run of 12 bases all of which have at least one C. I do not see tis but if you posted a normal sequence it would help a little. You do have to improve your sequencing either by a cleaner pcr product or a stronger sequencing signal as the quality is not good but that is not what you asked. Anyway my assessment is that this sample is not a heterozygote

https://seqcore.brcf.med.umich.edu/sites/default/files/.../interpret.html

Thanks but the link is not functioning ;)

Paul Rutland Thanks for valuable feed back and i attached a normal sequence for the same exone

Agree with Paul Rutland

regards

Thanks for valuable feedback and attached the normal sequence (at 490,TGGTGGC is present )

Hi Alshimaa

Thank you for the normal sequence which is an excellent sequence with base signal strengths varying from 1805 to 3890 so the signal to noise ratio is excellent. The run was analysed in march 2018 and all run parameters look good and almost the same as the sample sequence which was run in December 2017. Both runs start getting good sequence at about 2000 scans which is fine and indicates that the low signal strength of the 67f sample is not caused by too much salt in the sample causing poor loading. The signal strength of the bases in 67f is 84 to 285 with a high background and a signal/noise ratio of 11-23 which is poor. You might say that the low s/n ratio is normal for deletion sequences after the but in this case it does not start high then deteriorate after the deletion because there are 2 distinct sequences at every base .In your case the high background starts before the sequence and the second sequence under the main sequence is not equal intensity but much lower.. Also there are a few runs of the same base and with short deletions you usually see the upstream sequence readable under these bases and I cannot see this effect. I assume that your pcr negative control is negative and that the sequenced band was clean and strong . My feeling is that either the sequencing kit is getting old or that the ratio of sequencing primer to template is wrong ( not enough template perhaps) leading to a weak signal and the good sequence is simply becoming too weak to read with confidence where you think there may be a deletion

I would try sequencing from the other direction if you are not sure or possibly run a restriction digest ( CViKi-1 and LPNPI both cut your normal sequence twice close to the deletion area but only once when the deletion is present)

As another thought...sometimes when you pcr a heterozygote the pcr product forms a heteroduplex which runs as a second band running slower(larger) than the amplified template. Was there any sign of this effect on the agarose checker gel when you amplified the template?

. Best wishes,

Paul

Dear Paul

Thanks for valuable feedback and extremely detailed answer.

1-I attached the reverse sequence for the same sample and F&R of another sample with the same mutation , but was considered HOMO.

all data analysed with Geneious software.

2-For template /primer concentration, According to the company guidelines we send 12 µl of PCR product (at least 6ng/ µl -usually 12-20) and 3 µl of 10mM primer.

thank you for those files Alshimaa.,

some statistics attached

Both 67 and 19 samples look like homozygous deletion to me. The problem is the quality of the sequencing. Time is not an issue since 67 and 19 were run only a week apart so reagent integrity is unlikely to be an issue but some explanation of the file attached. The crl continuous read length for 14 is 611 bases and for the other files is 130-350.....not as good. Similarly the qv20 which is an indicator of the quality of the sequence in terms of signal /noise ratio of the bases is 624 bases in 14 but less than 360 for all other sequences. They are not as good as 14.

Taking randomly the intensity of the A bases called in 14 it is 1808 intensity and 59-117 in all other sequences....poor and getting down into the background region of random noise.

For some reason your sequencing has improved greatly and you need to look at the pcr and gels of 14 which works well and get the equivalent results for your research samples.

If you are only interested in the deletion then I suggest you do not sequence the whole amplimer of 600+ bases but buy 2 sequencing primers about 150 bases either side of the deletion and use these for sequencing and then with only a short sequence your signal strengths and ease of reading will be much higher and confidence greater.

bw

Paul

ps if you are really not sure about het/homo status then cloning and sequencing usually produces very clean sequences.

Dear Paul ,

Thank you very much for your interest and effort.

(For some reason your sequencing has improved greatly)

I revised my lab book and The primers used are not the same , I designed new primers later (but almost cover the same sequence) that is why I never faced that problem again :)

Thanks again for this lesson in Sanger sequencing .

Regards

Alshimaa