454 Sequencing Problems *Currently*

[Aniki]

Dear 454 users,

I have been having problems with 454 sequencing for a couple months now. I will outline all the problems I have encountered and hope you can weigh on with your suggestions or perhaps similiar issues (solutions). This is happening for both Lib-A and Lib-L. Although I seem to have better stats with Lib-L.

1. Sticky DNA Beads with Enrichment beads. Both DNA amplified and NULL beads will stick to Enrichment beads during washing/melting.

2. Sequencing runs have very low RAW WELL NUMBERS.

3. Very high rate of broken wells in SV titration reactions.

4. Severely low enrichment values (2-4%) in LV emPCR reactions despite using very high copies per bead numbers (>5cpb). Titrations seemed very good in duplicates but LV does not mimic SV titrations.

5. Sequencing runs yield much shorter read lengths than anticipated, despite the enrichment rate being between 10-15%.

If any of you have experienced any of these problems, please feel free to weigh on the issue and perhaps give me a solution. I am currently unable to resolve any of this.

Everything worked fine (well) until a couple months ago. Then suddently all these issues popped up. I am not doing anything different and I am using the same machines since the beginning.

Thank you.

[HMorrison]

Hi Aniki,

We have had some of these problems as well. We use LibA LV almost exclusively; for tag sequencing of large amplicons. We have elected not to upgrade to FLX+.

1. Sticky DNA Beads with Enrichment beads. Both DNA amplified and NULL beads will stick to Enrichment beads during washing/melting.

Definitely seen this; we have been able to resolve it by pre-annealing the enrichment primer to the beads. It was a brilliant discovery by one of the RAs.

2. Sequencing runs have very low RAW WELL NUMBERS.

Not so much. Generally, as long as our bead counts (Coulter Counter) are correct, we can load the ~1M per region desired.

3. Very high rate of broken wells in SV titration reactions.
4. Severely low enrichment values (2-4%) in LV emPCR reactions despite using very high copies per bead numbers (>5cpb). Titrations seemed very good in duplicates but LV does not mimic SV titrations

N/A

5. Sequencing runs yield much shorter read lengths than anticipated, despite the enrichment rate being between 10-15%.

Most definitely yes. We can, of course, run the shotgun processing pipeline, but then have to throw out most of the reads because they are too short or of poor quality. We need the bulk of the reads to be over 500 nt, and used to get them up until end of 2011. Huge loss due to 'read too short quality'.

Everything worked fine [well] until a couple months ago. Then suddently all these issues popped up. I am not doing anything different and I am using the same machines since the beginning.

That's exactly what we said--we are using the same protocol for amplification, clean up, and run that we always have (since Ti became available). Something has changed. However, Roche is working on the problem and has called in help from 454 team in Branford and apparently Jim Knight at the processing end. Changing various filters did not help, by the way, nor did endless bead washes.

Hilary Morrison

[Aniki]

Dear HMorrison,

thank you for your help. Can you please explain a little about how to pre-anneal the primers? I would love to know that as this way I can at least rule out of the issues.

Thank you very much.

[HMorrison]

pre-annealing enrichment primer

Hi,

This is the protocol (developed by Sharon Grim, who uses this forum but doesn't post often):

Combine 12.5 ul enrichment primer, 80 ul washed enrichment beads, 900 ul enhancing fluid. Anneal on the rotator for 5 min. This can be done in parallel with the melt step. After, wash 3x on the magnetic rack with 500-1000 ul enhancing fluid and resuspend in 80 ul volume again.

Problem is that it uses up enrichment primer over what is supplied in the kits.

[boerm]

sticky enrichment beads

We also encounter the same problem of sticky enrichment beads, we see the same results, short reads and high percentage dot and mixed reads, probably due to the null beads being present on the PTP plate.

We use the REMe unit for enrichment, and since this is a black box, you have no idea what happens, so it took a while before we decided to do the enrichment manually again, and yes the beads stick together and needed 25 washes instead of the programmed 11 washes on the REMe system. The recovery was much lower in the LV as calculated from the titration which was still done on the REMe system.

We have the upgrade on the 454 and use the plus kits, so here the problems are probably more extreme.

We will try preincubating the enrichment beads with the enrichment primer, thanks for the suggestion.

[lilyhu]

We have the same problem with Lib_L emPCR. Does anybody think this problem associated with different lot number of emPCR kit?

[boerm]

emPCR lot numbers

In our last 5 failed runs we used 3 different lot number of emPCR reagents, but the lot numbers of the enrichment beads were all the same, eg. 93866020 (emPCR recovery reagents)

[HMorrison]

We will try preincubating the enrichment beads with the enrichment primer, thanks for the suggestion.

So, did it help? We are still doing it. Current work-around is to underload the plate and simply expect to have to do two runs to get the number of reads that we routinely got up until Dec 2011. Been nearly a full year of this, now.

[yaximik]

I have exactly the same problems with GS Jr sine last spring - sticky beads, low numbers of raw wells, a lot of wells without the key, and a lot of short reads. As the number of control wells stays about the same and control reads are good, this got to be a emPCR issue. Sticky beads problem was easiest to resolve - amount of enrichment beads was empirically determined to be sufficient for less than 20% enrichment. That is, since beads are about 0.8 um in size, it takes a lot of them to hold strongly a much larger capture bead (~30 um) on the magnet. To many "positive" capture beads result in weak interaction making washes very frustrating. Positive means beads that have a complement to the enrichment oligo, which may come just from primer dimers or heterodimers. These are too short for sequencing, lack the key - that is where empty wells, short reads and no-key reads are likely coming from.

I used larger amounts of capture beads - any 0.8 um streptavidin magnetic beads work fine - and the problem of sticky beads gone. But all other downstream problems remained.

I think it is not the problem with the enrichment kit, but with the emPCR reagent kit. Somehow it favors formation of primer dimers/heterodimers screwing up everything downstream.

[lilyhu]

I have found the sticky beads was associated different lot of emulsion oil.

[yaximik]

Emulsion oil lot likely will affect stability of the emulsion. For example, I found that I get much better distribution of beads in droplets when I use different mixing conditions (I use IKA Turrax). The protocoll calls for 5 min/4000 rpm premixing and 5 min/2000 rpm after adding beads, but I found that a lot of droplets still contain multiple beads. 3000rpm/10 min or 2X 3000rpm/5min produced the majority of droplets with single beads, so this is what I am using. Yet this did not eliminate the downstream problems.

[lilyhu]

Emulsion oil lot likely will affect stability of the emulsion. For example, I found that I get much better distribution of beads in droplets when I use different mixing conditions (I use IKA Turrax). The protocoll calls for 5 min/4000 rpm premixing and 5 min/2000 rpm after adding beads, but I found that a lot of droplets still contain multiple beads. 3000rpm/10 min or 2X 3000rpm/5min produced the majority of droplets with single beads, so this is what I am using. Yet this did not eliminate the downstream problems.

How do you know the distribution of beads? thanks.

[yaximik]

Spread a drop of emulsion on the cover of 96-well plate or other plastic and look under inverted microscope with a phase contrast. One can easily see if the emulsion is stable, that is droplets are not merging.

[lilyhu]

Spread a drop of emulsion on the cover of 96-well plate or other plastic and look under inverted microscope with a phase contrast. One can easily see if the emulsion is stable, that is droplets are not merging.

Thanks a lot!

[exon]

We also suffered from low enrichment problem, mostly below 2%. The template that we are using is amplicon with fusion primer (with adaptor and barcode). We have checked the template with bioanalyzer and KAPA qPCR first. Then we used 1.7 cpb in the LV Lib-L emPCR. The enrichment is lower than 2% that we suspected the cpb not optimize, emPCR failed or the problem of the template? Then we went for SV using various cpb from 0.25 to 17, again the enrichment was <2% while the positive control (from previous work fine library) is OK but cpb increased from 1.7 to 14.6.