Roche Jr amplicon sequencing

[relaswar]

Has anyone attempted to sequence amplicons produced without Roche adapter sequences (only gene-specific primers)? Can they be sequenced by following standard shotgun protocol (perhaps w/o nebulisation)? Do I need to make any modifications to standard Roche Rapid Library protocol/emPCR protocol?

Thanks.

[MissDNA]

What you mean is use the RL protocol and kit to ligate the 454 adaptors to your PCR product instead of using the fusion primers, right?
I haven´t tried that yet but it is possible. You just have to pay attention to you product size. If your amplicon is smaller than ~500 bp chances are you will lose everything if you follow size selection as is described in the protocol. If your amplicon is longer than 1500 bp nebulization is recommended but you might need to adjust time/pressure.

[relaswar]

Thanks for your reply. Very helpful answer. My amplicon is ~500 bp, so I guess that's not an option in this instance.

[MissDNA]

It is an option but considering the size you may have to play with AMPure beads ratio or perhaps not use sizing solution that comes with the kit. I said you may end losing your amplicon because sizing selection is optmized to keep fragments within 600-900 bp and tends to eliminate everything that is bellow 500 bp.
I´ve never used Jr or looked into its protocols but I am assuming RL prep is the same as is for FLX, which is the instrument I work with.
Other thing you need to consider is the number of amplicons you have, since RL kits are expensive and worth for 12 reactions (usually you can do more than that but not much).

[relaswar]

Thanks again. However, in view of your further comment, I would like to expand my original query a little more.

The main reason for producing amplicons- without Roche adapters- is to have the freedom to use them on other platforms too. Regarding your point about size selection, I have got a query.

Would it be possible to optimise DNA:Bead ratio in such a way that fragments of >300 bp are retained (e.g. 0.7:1; BeadNA ratio) so that alll short fragments are removed? Therefater I could just continue with Adapater ligation etc. steps in the RL protocol. I agree with you about the cost, but can't think of any other way of doing this? Do you think this will work?Any more ideas? Thanks for your time.

[MissDNA]

If you have a 500bp amplicon the best platform to sequence it is 454.
It is possible to optimize ampure in order to remove fragments bellow 300 bp. The old General Library prep protocol called for an AMpure calibration in order to remove fragments bellow 350 bp. That protocol didn´t use sizing solution.
Have you read that Roche´s technical bulletin that gives you all the options for amplicon experiment design.
I don´t see why this approach would not work. This is an approach recomended by Roche.
Actually we´ve used something similar: one collaborator had synthesized cDNA using his own protocol, he sent it to us and we just followed RL protocol to ligate the adaptors. Sequencing worked really well.
I am out of other ideas, sorry.

[Jana667]

I have performed several successful runs on GS Jr using RL adaptors ligated on the PCR products. But my PCR products were 600 bp so I did not have to optimise AMPureBeads purification. But I am pretty sure that it is possible to optimise it for shorter PCR products.

[ajthomas]

I regularly sequence amplicons that are smaller than that. After amplification, I clean them up with AMPure beads using a ratio just below 1:1 (a little less beads than DNA), and that removes everything below about 200 or 250 bp. However, your lot of AMPure beads is most likely different that mine, so you might want to do some tests with your beads to see if that works for you too.

I would suggest, however, that if you're going to be sequencing this amplicon again, that you get a set of fusion primers (your primer sequence plus the sequencing adapter sequence). You can still generate the amplicon the way you are, then take a little of your PCR product (dilute it 1:100 or so) and run a second PCR for 10-15 cycles with the fusion primers to add the sequencing adapters to it. That will be a lot less expensive, more reliable, and easier than ligating adapters on afterward, and you can still sequence your amplicon on other platforms if you desire.

[relaswar]

I have got a query regarding recovery of amplicon product after purification with AMPure beads. I have repeatedly noticed that when I start with something like 25 ug total PCR product (amplicon) I usually end up with ~40 ng product only. I am very intrigued with high proportion of product loss just after one round of bead purification. I am following the protocol as suggested in Roche Junior manual. I have already optimised BeadNA ratio for removing short fragments (in my case it is 0.90:1 ratio), and is not a problem any more.

I wonder if this amount of product loss is common or is there something I am doing/not doing? Any help is appreciated. Thanks.

[sanju0891]

Originally Posted by relaswar

I have got a query regarding recovery of amplicon product after purification with AMPure beads. I have repeatedly noticed that when I start with something like 25 ug total PCR product (amplicon) I usually end up with ~40 ng product only. I am very intrigued with high proportion of product loss just after one round of bead purification. I am following the protocol as suggested in Roche Junior manual. I have already optimised BeadNA ratio for removing short fragments (in my case it is 0.90:1 ratio), and is not a problem any more.

I wonder if this amount of product loss is common or is there something I am doing/not doing? Any help is appreciated. Thanks.

Even we had the same problem. we had to remove below 250bp so we did a 3 step purification
1. 1.8:1 ratio which removed all below 100bp and the conc was around 40-50ng/µl
2. 0.8:1 ratio which faintly removed all below 250 bp and the conc was drastcilly reduced to 4-10ng/µl
3. 0.8:1 ration which completely removed all the bands below 250bp but the conc was not able to be quantified. the nanodrop shows values in negative conc (we know it is not so sensitive but still we gave a try )

we seriously need to remove evrythng below 250bp to get high number of reads but if we do a ampure purification twice our conc is going too low. we are not able to solve this.

any suggestions. we were thinking to use the picogreen or qpcr for quantitation. but im not sure even this helps

[Jana667]

I think, that using picogreen or qpcr might help, I use routinely both for DNA quantification...these methods are much more sensitive and accurate than nanodrop. For emPCR you need only a small amount of DNA...

[ParkerKB]

To do as suggested by ajthomas you need to do a 2-step PCR--first amp uses the locus-specific primer combined with a "universal tail" (often an M13 sequence), the second amp primes the "universal tail" and attaches the 454 sequences. The product of your first PCR would not have 454 sequences, but would have the universal tail. I don't know if/how this would affect applying it to other platforms.

See the "Guidelines for Amplicon Experimental Design" section 4, and the attached pubs.

[nexgengirl]

You can also skip using the sizing solution. Check out the attached paper for further details on amplicon sequencing with 454 junior and ligating adaptors