How to interprete direct sequencing results? - (May/19/2007 )
For the BSP-direct sequencing I normally use twice the normal concentration (for the Kit I use, 3.4pM primer is suggested, I use 6.4pM). It may be worth a try? But I think you may need more amplicon - do you quantify them? I regularly use 20ng of the purified amplicon (that's again about twice the suggested amount for my kit).
I am not too sure, as the most irregular G's occur only in the A lanes. That is more indicative of incomplete conversion, but may look worse due to the software bias. Maybe you think about C/G tagged primers, too? It's tested in a paper by the Spivak group, and I have made very good experiences with that.
I always check the concentration after gel-purification with 3ul Probe versus 3ul DNA-ladder (concentr. 100ng/ul) on a agarose gel and the probe -band is always MUCH stronger as the bands of the ladder.. (I always concentrate my PCR-product with Ethanol from 75ul to 15ul for gel extraktion, so I get always a high concentrated solution) ...
Intreastingly, the first time i sequenced my samples I had really low concentrations (the bands were hardly visible on the gel) but I got similar signals as now...(?)
...I really can´t explain those low sequencing -signals! also I asked about the concentration in the sequencing -lab, and they told me that the conc. was in all samples ok, also they acknowledged the fact that those samples were much more concentrate as the first ones....
If I may ask you one more question: like you can see in the attachment in my previous insert, the peaksignal decrease continuously and especially after Base ~140-150. Is this normal?
That's absolutely normal, I experience the same.
Low sequencnig signals - if you get the same results with a very low concentration, maybe you now have too much template? As the extension reaction is a linear (non-exponentiell) reaction, the correct primer:template ratio is crucial. It may be worth doing some dilution series with different tamplate and primer concentrations (like for optimizing the PCR). Doing so you could figure out the optimal concentrations and maybe solve the problem with the low intensities?
...yes, it sounds good, maybe a template-dilution and an increase in the primerconc., let try it!
Thanks for the help!
Just let us know, when (and how) you solved the problem.
I discussed about direct sequencing after BSP with ABI -system with a prof. and he recommended me to verify the ABI-results from direct sequencing with a cloning approach. What is your opinion about that?
There are some papers reporting on that and they find a good correlation. The problem with cloning is, that you need to sequence a lot of clones to get results that are statistically reliable (ten clones are good for unmethylated vs. methylated, if you want a measurement ten percent-wise, you probably need to sequence qround 50 clones).
I have tried to avoid it and so far succeeded with this strategy...
Sequenom Epityper array anaylsis is a variation to direct sequencing and they have shown good correlation with cloning and sequencing, as Krumel stated, you would need to sequence quite a few clones to verify the direct sequencing results. The number you select for analysis is arbitrary.
Hi methylation kings !
I'm new in this field. I would like to determine the methylation status of about 120 CpG, into the promoter of my gene of interest. There is very few data about it in the literature. The bisulfite conversion was done with the EZ DNA Methylation Kit from Zymo Research. I managed to amplify this promoter with 2 PCR (475 and 1095 pb), using primers designed with the help of MethylPrimerExpress. After Qiagen purification, I have clear bands, but no very intense, even after 45 cycles.
I have designed 9 sequence reactions (3 for the 1st PCR, 6 for the 2nd) to read almost the entire sequence of the promoter. I use Applied BigDye Terminator V3.1 and a sepharose purification method, on plaques. We, at the lab, have two sequencers : an Applied 3100 with ABI basecaller, and a 3130 with KB basecaller.
I tried to read my sequence on this two sequencers, but for most of my sequences, I get a noisy background : C for forward sequences and G for reverse sequences. An exemple is attached, it is the same sequence reaction. The 3130 sequence is on the top, the other is the 3100 sequence. The sequence was read with a reverse primer, so I inversed/complemented it to improve the reading of potential CpGs. As you can see, I globally get low signals, so I will try to enhance them. According to the previous posts, I also think of a problem of weak C or G signal compensation by both basecalling softwares.
So, does anybody know a way to limit/stop this compensation ? An option to choose during the acquisition of data or during the analysis ?
PS : I plan to sequence about 60 different gDNA, so I think cloning is not an option, at this time.
Thanks in advance for your help.
Have you considered a nested PCR strategy? I have so far never yielded enough amplicon for sequencing using only one round of PCR.
To reduce the C/G noise you can consider the use of C/G enriched primers for the second round PCR. There is a paper by the Spivack group (Han et al.) and some topics in this forum also.