[ajthomas]

The truncated oligos might or might not bind to the beads or the amplification primer, depending on where they're truncated. Where this might be a problem is if the primer with one MID has significantly more or fewer truncated oligos than the others. In that case, the balance of reads you get out may be a little off from what you desire and put into the system. But yes, once the molecule binds and goes through the first round or two of amplification, the molecules will be full length. So as long as a particular molecule isn't truncated at one end or the other far enough from the end that it won't bind, it will be fine.

By the time you add the sequencing primer, the molecules will be full length, so that won't be a problem.

The bead enrichment step works by attaching a biotinylated oligo that matches the adapter that is not attached to the bead. The biotin is used to separate the DNA-containing beads from those with no DNA (hence "enrichment"). Ideally, you only want about 5-20% of the beads or so to have DNA on them, because if you have more than that you will have a lot of mixed reads.

[eladSeq]

So I have designed sets of A and B fusion primers with varying barcodes to get the desired offsetting of the flow reads. This will require 42 primers to make and pool 21 amplicons. Because of the number of primers needed, we have decided to go with ordering regular desalted oligos rather than spending a lot of money on purified. I have come across some researchers who use desalted and some that use purified, but no side-by-side comparison whether there is a difference.

Some brainstorming on the effect of oligo purity and questions I have about the emPCR steps in general:

At this oligo length (54-59 bases) IDT says that about 65% of the synthesized oligos will be full length. So would the 5' truncated products cause a problem? The PCRs should work fine since the 3' ends would be good. Perhaps 5' truncated amplicons would bind less efficiently to the capture beads, but the titration step should account for this and determine the optimal DNA loading quantity, right?

Would there be an issue with the sequencing primer annealing? I'm not sure how this part works. Does the sequencing primer anneal to the A or B adaptor sequence?

Also, I am unclear as to how the bead enrichment step works and what this actually does, if anybody could help me understand this?

Sorry about all the questions, but I am hoping to try and get this library prepared correctly the first time, and avoid problems down the road. Thanks all.

[ajthomas]

That's an intriguing idea. It seems to me that should help. If you're going to do it, you should carefully pick your MIDs so as to maximize the effect. After reading this, curiosity got the better of me and I looked at the MIDs I use to see how much effect they may have. I use the 48 MIDs that Fluidigm recommends for their Access Array system, which are mostly just Roche's first 50 MIDs with a few replaced that I assume they found didn't work well for one reason or another. Here's what I found:

flows # of MIDs
14 1
15 3
16 6
17 22
18 9
19 3
20 4

Nearly half of them require will end on the same flow, so if you try this route, be selective in which MIDs you choose.

[eladSeq]

Amplicon sequencing not supported on FLX+

Thank you everybody for your input.

Indeed, FLX+ is not currently supported for amplicons. From my understanding, there would be too much "flashing" due to the homogeneity of the sequences, such that SNPs would get obscured.

So I'm planning to use the regular titanium chemistry and split my gene into 3 amplicons, that can be covered by the 400 base reads.

I was informed that adding multiple (10 or more) different barcodes for each amplicon would help add complexity to the mixture such that bases are "offset" in the flowgram and reduce amount of "flashing". Has anybody tried this or recommend it? When I spoke to Roche rep he said that didn't have any data about it, and that it wouldn't make a difference if using MIDs since they are all the same length. However, it seems to me that the sequence matters, since the nucleotides are added in a specific order.

For example, supposing bases are added in the following order: T A C G
A barcode of CTG would take 8 nucleotide flows to complete, while a barcode of GAC would take 7 nucleotide flows, thereby offsetting the start of the desired sequence.

Thanks,
Elad

[MissDNA]

I think Roche decided to do the first upgrade here in Brazil in that other lab, because they have two GS-FLX, which is not our case.
It was indeed some software problem and now they are waiting for some installation CD, in order to complete the process. Since things take a lot longer around here, it will take awhile.
Biggest problem is we already ordered 7 FLX+ kits, and they are supposed to arrive in January...

For the people who already have the upgrade: are the runs using Titanium still good?

[tokikake]

Something regarding to the equipment temperature, which made me think about some post I read here mentioning temperature problems. At this point, we are a bit scared of the upgrade since our machine and sotware are running really well as is.

That is very interesting! I wrote some weeks ago about our problems with the upgrade and last week we had a visit from the service and they replaced the PTP heater because of some temp problems...I heard about the same problems from another sequencing lab. Honestly, after all the troubles in the last weeks I would definitely wait with the upgrade.

[RCJK]

Some feedback from 454 re: amplicon processing after sequencing with XL+ kits:

Amplicon processing for this dataset can be done as runAnalysisPipeAmplicons like every other dataset. However, since long Amplicons are not supported, the software (as of v2.6) as it currently stands is not tuned to expect long Amplicons. Consequently, the pipeline along with parameters, thresholds, error checkpoints and options are not modified to reflect this and correct results may not be obtained. So, while it is possible to run the pipeline and get reads results may not all be of good quality. Please keep in mind that the results and / or subsequent comparisons may not be accurate.


What I am unsure of after this response is whether or not "short" or <600bp amplicons sequenced with the XL+ kit and processed with amplicon signal processing as previously done would/should be ok/accurate. I'm awaiting clarification on this, but not holding my breath for a straight answer.
The amplicons I mentioned a few posts above were short (~230bp-480, but mostly <300bp) and were emPCRd using the short amplicon protocols.
Otherwise, the shotgun libraries have been looking really good with modal read lengths of ~700-800bp. So apart from the first failed run, I'd say the rest have been successful.

[MissDNA]

We just found out our upgrade won´t happen till January, also there was some software problem with some upgrade that has been made around here. Something regarding to the equipment temperature, which made me think about some post I read here mentioning temperature problems. At this point, we are a bit scared of the upgrade since our machine and sotware are running really well as is.

We are purchasing an Ion Torrent, that hopefully will be installed begining of next year. We decided in favor of having an Ion before we knew about MiSeq. Even though I never worked with Illumina an know very little about their technology, I really think it could have been a better purchase. We will see.

[pmiguel]

About PE, I don´t see the benefit of running it with a FLX+ since the library prep protocol was not modified, so the libraries have still shorter fragments than the desired for a good FLX+ run. I really wonder if Roche is working on optimizing PE protocols since they are already obsolete and time consuming.

You could tweak the shearing parameters for the PE protocol to favor longer inserts.

I would not hold my breathe for FLX+ protocols. I think much of the research effort given to the GS-FLX shifted to the FLX Jr as Illumina sucked up more and more of the next gen market place. This is what I think caused the 1-2 year delay in the release of FLX+ chemistry. Big mistake, in my opinion, because MiSeqs and IonTorrents will probably completely crush any burgeoning opportunities for Roche to gain a strong foothold in the desktop nextgen sequencer market. Whereas having FLX+ in place 1-2 years earlier might well have solidified the 454 niche in core labs.

If Roche Applied Sciences can hold on for a while longer, they may begin to see some pick up in sales as the heavily "early adopter" phase of next gen sequencing matures into something more mainstream. That process would be really helped by run processing software that did reasonable analysis of failure that could be used by cores to rapidly identify where issues arose. For example, currently you have to upload a run to tech support to get a global assessment of signal droop across a run. As a result Roche tech support has to waste a larger percentage of their time trying to troubleshoot issues that would be trivial for a core to deal with directly.

--
Phillip

[pmiguel]

XL+ is still not recommended for amplicons. Another lab that has run them has said that they have had consistently worse results than when the same amplicons were run with the XLR70. Roche has lowered the pricing of my XLR70 kits to be the same as the XL+ kits so that I'll use them for amplicons. That said, I did just do 1/8th of amplicons on an XL+ run and am waiting for feedback from the customer. Also, I'm not sure if you need to do anything different for amplicon signal processing with the XL+ kits. With shotgun processing I had ~85% pass filter, then when I did amplicon processing, it went down to ~12%. With modifying the filters to trim from the 3' end instead of discarding the reads, it went up to ~18% pass filter. Also, the controls looked weird and gave really long read lengths (1-2KB) after amplicon processing, so I'm not sure what that's all about.

See also this thread:

http://seqanswers.com/forums/showthread.php?t=15838

Seems to imply that even using XLR70 kits, there may be issues with the really long read lengths if the instrument is FLX+.

Caveat being that Indiana may have modified their processing pipeline from Roche standards. I don't know.

--
Phillip

[MissDNA]

About PE, I don´t see the benefit of running it with a FLX+ since the library prep protocol was not modified, so the libraries have still shorter fragments than the desired for a good FLX+ run. I really wonder if Roche is working on optimizing PE protocols since they are already obsolete and time consuming.

[madseq]

In principle, it is not possible to perform a XL+ run (400 cycles) with amplicons because there is no an Amplicon pipeline for 400 cycle runs. It is not possible to analyze an amplicon library properly after the run.

However...
For amplicon and shotgun combination in the same plate I think that it could somehow be possible to modify the .cwf files by deleting the images corresponding to the last 200 cycles and then try the amplicon pipeline. It is not very good idea to use shotgun analysis for amplicons. The Amplicon pipeline takes into account the fact that the sample will be of low diversitity, which means that a lot of wells will have the same signal at the same time.

Below you´ll find what I was told from customer support when I asked about this issue:


- FLX+ chemistry is not compatible with Amplicon sequencing
- FLX+ instruments are backwards compatible to Titanium chemistry, so you can still do Amplicon sequencing using a Titanium kit. Limitation is the pipeline, as SW 2.6 does not contain an Amplicon pipeline for 400 cycle runs
- FLX+ sequencing kit and Titanium sequencing kit is different- FLX+ runscript is 400 cycles, Titanium runscript is 200 cycles. You cannot start a 200 cycle run using a FLX+ kit
- Titanium (shotgun and paired end, NOT amplicon) can be run using a FLX+ kit without a benefit in readlength

[ajthomas]

I've heard that from a couple of sources. In my case, my libraries consist of 6 different amplicons with very high polymorphism. That means that the strong synchronous signals normally seen with amplicons that make sequencing them more difficult for the GS FLX should be reduced compared to a lot of other amplicon projects. I'm hopeful that means it might work a little better for me. I hope it does because two of my amplicons are adjacent exons in a gene that I would really rather sequence together rather than separately. The two exons with the intervening intron is short enough that I'm only about 50bp short of sequencing it reliably with the FLX. I'm hopeful that I might be able to get those additional 50bp by using the FLX+. If not, oh well, I guess I'll have to wait for improvements to the system/reagents to allow it to work.

[RCJK]

XL+ is still not recommended for amplicons. Another lab that has run them has said that they have had consistently worse results than when the same amplicons were run with the XLR70. Roche has lowered the pricing of my XLR70 kits to be the same as the XL+ kits so that I'll use them for amplicons. That said, I did just do 1/8th of amplicons on an XL+ run and am waiting for feedback from the customer. Also, I'm not sure if you need to do anything different for amplicon signal processing with the XL+ kits. With shotgun processing I had ~85% pass filter, then when I did amplicon processing, it went down to ~12%. With modifying the filters to trim from the 3' end instead of discarding the reads, it went up to ~18% pass filter. Also, the controls looked weird and gave really long read lengths (1-2KB) after amplicon processing, so I'm not sure what that's all about.

[ajthomas]

I haven't heard anything different concerning amplicons and FLX+ chemistry either. Saying that, however, I'm going to give it a try some time in the next few weeks. Not an entire run, mind you, just a couple of sections of a plate on which I'm running some other stuff. I'll try to remember to share my findings here.