First sequencing results are terrible

[Dakota Hamill]

Well in the spirit of sharing is caring  I wanted to share the first 4 samples I had sequenced.  I'd also like to try to get some tips from anyone who does this more often.  No priming happened on the first run so there was basically nothing to be read and the company re-tried for free to try to get it to pass QA.  The number of bases identified increased, but it still was pretty bad.

I've uploaded 8 files, the first 4 are Trace files which show the red/green/blue/black peaks for the four different nucleotides.  The second four are Seq files, which is basically a gen bank file or word text file, just a list of GTAC and a lot of N's in this case!  It can be opened in most DNA utility tool programs.

Here is a screen shot of the poor priming/no priming with the correlated confidence score they give.  http://gyazo.com/b9aa84842e878b97e284c4ba8b2276d9

NSI1 (forward) GAT TGA ATG GCT TAG TGA GG
NLB4 (reverse) GGA TTC TCA CCC TCT ATG AC
ITS1 (forward) TCC GTA GGT GAA CCT GCG G
ITS4 (reverse) TCC TCC GCT TAT TGA TAT GC

There are the primers.

In this particular case, there are perhaps to many potential variables.  My friend prepped the DNA and primers himself, using a nanodrop to try to get a DNA concentration.  He said, for all he knew he could have forgotten something completely.  Secondly, they were sent out on a Thursday or Friday, meaning they sat all weekend and weren't sampled until Tues/Wed which equals days at room temperature about, in just H2O, which could have lead to chopped up and degraded DNA.  

In most cases I've read the forward primer is a first go-to in terms of a sequencing primer...can this generally be considered the case?  Would a universal primer be better?

Do you often send your DNA/primers in TE or H2O?  They've mentioned TE can screw up sequencing via Mg2++ chelation.

Do you often do forward + reverse sequence reads for better confidence?  I'd generally pay for a forward/reverse but for this control with a known species, we wanted to see if just the forward primer would work, and we had confidence it would work just because these primers are so well documented and used.

Thanks!

[Matt Lawes]

Looking at your sequencing primers, lots of GC at the 3 prime ends may be causing a bunch of mispriming / random priming .... that would give the 'lot of Ns'.

Anyone else see this as a problem?

>matt

Sent from my Verizon Wireless 4G LTE DROID

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[Dakota Hamill]

Well for the ITS1 F primer I can see that, but not so much for the others.  I'm glad you pointed it out though because it is something to consider in the future.  I actually had some other primers synthesized and was trying to avoid GC rich areas as well as triple base repeats in the 3' end and luckily was able to use an R nucleotide to get G or A I believe.   I ordered a second set of primers with an M13 phage sequence included so we'll see how that goes because it's a common sequencing plasmid.  

I just finished a PCR rxn of 3 fungal samples an hour ago and put them on ice.  When I can verify amplification on Thursday I might send fedex overnight this time and get them sequences asap and see if it helps.   Thanks for the comment.

[Jeswin]

On Tue, Feb 12, 2013 at 9:03 PM, Dakota Hamill <dko...@gmail.com> wrote:
>
> In this particular case, there are perhaps to many potential variables. My
> friend prepped the DNA and primers himself, using a nanodrop to try to get a
> DNA concentration. He said, for all he knew he could have forgotten

What are the requirements for concentration? I used Eurofins and for
sequencing PCR, they need 8uL of 20-40 ng DNA with 4uL of 2uM primer.
Our primers are at 10uM so I just add 0.8 uL primer. I have
successfully sent DNA with concentrations as low as 9 or 10 ng. It's
best to make sure that primers were added in the first place. It can
happen; I have switched fwd and rev primers before.

> something completely. Secondly, they were sent out on a Thursday or Friday,
> meaning they sat all weekend and weren't sampled until Tues/Wed which equals
> days at room temperature about, in just H2O, which could have lead to
> chopped up and degraded DNA.

It is not recommended that DNA be shipped over the weekend.

>
> In most cases I've read the forward primer is a first go-to in terms of a
> sequencing primer...can this generally be considered the case? Would a
> universal primer be better?
>

What is a universal primer?

> Do you often send your DNA/primers in TE or H2O? They've mentioned TE can
> screw up sequencing via Mg2++ chelation.
>

Yes, I have heard that too but it was only last week I learned that.
Previously, I had sent samples eluted with TE buffer without a
problem. Now I elute my DNA with Qiagen Buffer EB (Tris-HCl). My
samples are sent with the DNA, primer, and up to 12 uL with H2O. Never
had a problem. Also, Nanodrop concentrations are probably not reliable
for readings below 10-15 ng.

> Do you often do forward + reverse sequence reads for better confidence? I'd
> generally pay for a forward/reverse but for this control with a known
> species, we wanted to see if just the forward primer would work, and we had
> confidence it would work just because these primers are so well documented
> and used.
>

In some cases, we run both primers. The primers I used have been used
in PCR so I have some confidence that they are working.

Were you sequencing a PCR or extracted DNA? Maybe you need to PCR
amplify before you sequence.

[Dakota Hamill]

Thanks Jeswin for the thought out detailed answer!  We were sequencing PCR products, and based on the brightness of the gel I had guessed we got about 300ng of product. (total, not just from the 5uL gel sample)  Based on the nanodrop my friend said we got about 500-650ng in some cases I believe (25uL total, but 5uL to the gel, kept 20uL for sequencing, 10ul for forward, 10ul for reverse if needed).  So I'm afraid we put to much DNA in.  He tried to get them down to 50ng about, and we sent primers at 5uM concentration.  We managed to get everything to basically what they requested, but it was un-cleaned PCR product, and we had 2 free reactions for custom orders, and assumed GeneWiz would clean them up.  So, given that the reaction was still "dirty" and filled with salts, enzymes etc and was left for 4-5 days, I'm guessing it just got so chopped up or degraded it gave really poor results.

I did 3 PCR rxns last night and will image them on a gel Thursday, and if all goes well, FedX them right out or drop them off in Cambridge if necessary!  Hopefully that should help.

[Tom Randall]

My experience has been sending my DNA/primers to a local University and having them do the sequencing. They have suggested guidelines for submission (amount of DNA/primer to send) best ways to prep plasmids or PCR fragments, etc. These guidelines can differ a lot depending on who is doing the sequencing. Did your friend follow the suggestions of the institution doing the sequencing, or were these even available?

Also, I think the idea of the reagants sitting around in dH2O could have been a serious problem due to the potential degradation as you suggest. I have had issues when I submitted in TE, so dH2O is good, but keep samples frozen/cold as much as possible.

[Josiah Zayner]

DNA is very stable. Very very stable. This is why we can sequence Wooly Mammoth genes.
DNA doesn't degrade so easy and especially not just over a weekend. This paper says the rate of spontaneous hydrolysis of a phosphodiester linkage is ~10^-13 / sec which is a halflife of 140,000 years.

Nanodropping a non-cleaned PCR reaction doesn't really work. In fact the results are probably really off.

Are there lots of bands in your gel?  Normally one doesn't send a PCR reaction to be sequenced. They will cut out the band on the gel and then purify the DNA from the band on the gel to have a homogenous sample

I have sent thousands of samples for sequencing and the three most common reasons for having bad sequencing results are:
1. Not enough DNA
2. Not clean DNA
3. Not homogenous DNA

It seems obvious from your forward primer electropherograms make it seem obvious that there is no sample or your primer is not working because there is no signal. Or your sample is just so nonhomogenous you are not having any consistent reads.

Forward, reverse doesn't really matter it is just that forward are easier to interpret.

It is strange that only the reverse works? Lots of strange stuff can happen on sequencing I know on the big dye machine that I usually send my stuff to they have bleed through and bunch of weird issues when one doesn't have much product.

Doing Blast on the reverse sequences it looks like they are from a cloning vector. Any idea about this? Template? Contamination?

[Nathan McCorkle]

On Thu, Feb 14, 2013 at 3:51 PM, Josiah Zayner <josiah...@gmail.com> wrote:
> DNA is very stable. Very very stable. This is why we can sequence Wooly
> Mammoth genes.

aren't the specimens generally fossilized or found in permafrost (frozen)?

> DNA doesn't degrade so easy and especially not just over a weekend.

I've had it happen before

> This
> paper says the rate of spontaneous hydrolysis of a phosphodiester linkage is
> ~10^-13 / sec which is a halflife of 140,000 years.
>

not due to decay, due to nuclease contamination

--
-Nathan

[Nathan McCorkle]

On Thu, Feb 14, 2013 at 4:22 PM, Nathan McCorkle <nmz...@gmail.com> wrote:

>
> not due to decay, due to nuclease contamination
>

(poor lab skill/practice)

> --
> -Nathan



--
-Nathan

[Josiah Zayner]

Sure the specimens are frozen but RNA and usually most all protein are degraded.

DNA degradation does happen, I am not saying it doesn't. What I am saying is that the probability that the problem was DNA degradation is tiny. 

I work without gloves, rarely clean my pipettes or lab bench and I have never had a problem with DNA nuclease contamination. It is not a common problem. Again it could be possible but not a common problem.

This Dakota person seems competent so I am assuming it is something else. However, I am just making a guess based on the information she provided.

[Jeswin]

On Thu, Feb 14, 2013 at 6:51 PM, Josiah Zayner <josiah...@gmail.com> wrote:
>
> Nanodropping a non-cleaned PCR reaction doesn't really work. In fact the
> results are probably really off.
>
> Are there lots of bands in your gel? Normally one doesn't send a PCR
> reaction to be sequenced. They will cut out the band on the gel and then
> purify the DNA from the band on the gel to have a homogenous sample
>

Oh, is that what Dakota did? I read that he ran a gel but I thought
the band was cut and purified. I have never measure concentration or
sequenced PCR products that were not purified from gel
electrophoresis. This might be the problem.

[Dakota Hamill]

Here is the gel picture.

http://basementbiotech.org/wp-content/uploads/2012/12/b73e8c7eb29e45b87196fee9fbd86afa.png

The lane to the left and right of the left-most ladder was sent for sequencing.  NOT the excised gel, but the unpurified PCR products.  It was a custom order specifying these were un-purified PCR products.

I'm guessing my friend just completely forgot to add the DNA or a primer, which after speaking to him, seems like a likely event.

Only the forward primers were used, but I assume that when they did the second attempts because it failed quality controlled, they used their own sequencing primers, which is where you are getting the BLAST hit from.

And these were the Universal Primers I was speaking of earlier Jeswin.

http://www.genewiz.com/public/universalprimers.aspx

 I am just making a guess based on the information she provided.

Also, I'm probably the ugliest woman you'll ever meet.

Thanks for all the discussion on the topic, I've learned a few more things and will hopefully get quality data next week if the repeat reactions I did two days ago worked.

[Dakota Hamill]

Appreciate the detailed response.

The new set of primers I had made included the M13 phage primer set to be used in the plasmid for sequencing.  The fungal ones I used, ITS and NSI, did not include that.  Based on the fact that no priming even happened in the original reaction I have a feeling the primers weren't even in there in the first place, but I don't know.   ITS and NSI are the most common used fungal barcoding primers so I have confidence it is not the primers design that is at fault, but most likely user error in this case.  I do have a PCR cleanup kit I didn't use this time, so I'll just do it again.   I can only guestimate DNA concentration because I don't have access to a UV spec or nanodrop regularly.  And this time I'll be excising the gel band and sending that probably for sequencing.

[Nathan McCorkle]

On Fri, Feb 15, 2013 at 10:22 AM, Dakota Hamill <dko...@gmail.com> wrote:
> I can only
> guestimate DNA concentration because I don't have access to a UV spec or
> nanodrop regularly. And this time I'll be excising the gel band and sending
> that probably for sequencing.

what kind of dye are you using, the concentrations of DNA are pretty
well correlated with dye intensity... for instance gelGreen says some
of their customers have reported 0.1ng DNA detection limit... but this
doesn't really mean much because they don't give an area (what the
camera would see) or a depth (how many molecules are behind a camera
pixel)... but if you assume a standard 50mL gel, you can probably get
a decent guesstimate.
--
-Nathan