short reads from amplicon for 454 using Titanium chemistry

[pseudorabies]

Recently, I did amplicon re-sequencing project to test variation in my barcoded samples. The desgined primers pairs for amplicon is little bit ambitious with length ~690bp for the amplicon, it was supposed that 454 using Titanium chemistry will give a mean 400bp reads. It turned out only a mean length of 225bp in my data, which make me real frustration. A lot of short reads can not assembly in my data. As I have high coverage of data and it gave the complete assembly for these amplicons, but I really don not know why it gives such a short reads. Dose anyone have the same experience? Any troubleshooting for experiment? Any comments?

[pmiguel]

There is a new protocol available on the 454.com/my454 website that gives a lot of guidance on amplicon related issues. One recommendation for amplicons of large size is to modify the amplification mix and the thermal cycler program.

For your current data set, there are some parameters you can change in a local parameter xml file before re-running the filter analysis pipeline. Definitely worth a shot.

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Phillip

[pseudorabies]

Could you give me some links, I can only find the 2006 protocol. Thanks!

[RCJK]

He did. The link is: 454.com/my454
Have a look at the new application guide "454 Sequencing System Guidelines for Amplicon Experimental Design (May 2011)". For an amplicon of 690bp it would be recommended to try the long amplicon conditions described.

[suefo]

Take a look at TCB 2011-001. I spoke with 454 this week about this issue and they recomended this bulletin. Even though it reads for Lib-A, 454 felt it would also work with Lib-L. BUT you will need to adjust the amount of primer and amp mix to be proportional for a Lib-L. We were hoping to actually run the sequence last night, but the e-box went on our Junior (that's the communication computer inside the Junior) and we had to abort. Waiting on a service call now.

[pmiguel]

How about just using the gsflx+ emPCR protocols? They are for Lib-L and use the same thermal cycler program as specified in the long amplicon protocol.

Again, the manuals are all on 454.com/my454. However, if you don't get a response to the registration request in a day or two, I would suggest contacting your sales rep to expedite registration. Without doing that, my experience is that registration never happens.

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Phillip

[pseudorabies]

Thanks for your message, I do try 454.com/my454, but I still can not get in probably because I not the people who buy the machine (registration does never happen!). Can anyone send me a copy of that "454 Sequencing System Guidelines for Amplicon Experimental Design (May 2011)" AND "TCB 2011-001"? Thanks a lot.
[email protected]

[suefo]

I will send them to you. You should be able to see them yourself. They should be under DOCUMENTS on the my454.com/my454 window. The last item listed in that drop down menu is Technical Bulletins. I'm using my home computer (not registered) I can see them.

[pmiguel]

Hi suefo,
No, your home computer must have your credentials cached. You must be registered to access this site.
It is actually extremely irritating. The registration process is not automated. Not sure what the psuedo secrecy is meant to accomplish.

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Phillip

[proteasome]

I've done 454 FLX and Jr sequencing on 1,000 bp amplicons without modifying the protocol at all and not had problems. Have you checked that there's not some other problem going on with your template that's causing you to get short reads? I once had an area of homology between one of the adapter sequences and a region of my target which led to short products being generated during emPCR. A way to test this is to try Lib-L sequencing your amplicons with different adapters (although they will only be unidirectional unless you swap adapters and do two PCRs per amplicon). Roche has a technical bulletin on how to do this.