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Old 04-18-2013, 03:56 AM   #21
JackieBadger
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I'm just wondering what studies/genotyping approaches people are using in the context of this discussion?
We also see carry over between amplicons....we have mainly attributed this to lab sample set up contamination (as we also saw it in some previous 454 work). There may well be some MiSeq run carry over too...however this level of contamination is easily identified vs true loci due to the large depths we attain... I could see how it would become as issue when using low coverage to genotype, but plenty of studies recommend increasing depth to facilitate genotyping ease. This seems a little more hassle free than investing in more barcodes....

In addition I guess this problem is more severe when the machine is being run multiple times in one week, as a wash is required ever seven days.
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Old 04-18-2013, 06:23 AM   #22
james hadfield
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And when we start with 2500 I think we'll use cBot clustering for samples where we want to keep comtamination to an absolute minimum.

The main problem from a cancer perspective is tumour heterogeneity and labs trying to understand if minor clones are important. The earlier we can detect them (by loking at mutatnt allele frequency) the better. And this is likley to need sub 1% detection levels.
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Old 04-18-2013, 07:08 AM   #23
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Quote:
Originally Posted by james hadfield View Post
And when we start with 2500 I think we'll use cBot clustering for samples where we want to keep comtamination to an absolute minimum.

The main problem from a cancer perspective is tumour heterogeneity and labs trying to understand if minor clones are important. The earlier we can detect them (by loking at mutatnt allele frequency) the better. And this is likley to need sub 1% detection levels.
Rapid chemistry does not allow cBot clustering. Even with the "duo" kit all you get is first strand synthesis. The actual bridge PCR happens on the HiSeq. So I am not sure where that leaves you. Might solve the problem, or not.

But, just because this is a problem on the MiSeq does not mean it will be on the HiSeq. Enough of the right type of washes and the issue can be driven to arbitrarily low levels.

Has anyone done any back-to-back Rapid HiSeq runs that could be used to test for previous template contamination?
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Old 04-18-2013, 11:12 AM   #24
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The duo kit on the HiSeq might be enough to overcome the problem with carry-over since no template ever enters the HiSeq's fluid lines. I wonder if a couple of extra manual washes at just the template position would be enough to help resolve the problem on the MiSeq.
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Old 04-22-2013, 04:15 AM   #25
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We've also seen a carry-over of 0,01% to 0,1% between runs, depending on the washes that were done in between (just post- or also maintenance wash, and how many times).
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Old 04-22-2013, 08:44 AM   #26
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I picked three adjacent runs with highly dissimilar samples:
09 Apr 13 with a mix of 3 well-annotated and sequenced bacterial genomes
10 Apr 13 with all non-bacteria stuff (broad mix of other stuff)
17 Apr 13 with arabidopsis.

I just took the "undetermined" bins from the 10Apr and 17Apr runs and mapped to the bacterial genomes (I didn't check to try to sort out if indexes were repeated, etc - that could be an independent check but I'm just scouting at the moment).

10Apr shows 0.027% bacterial genome sequences in the Undetermined bin (% mapping divided by total reads from the run) and 17Apr shows 0.024% bacterial genome sequences. Now some of these may be phiX control sequences that happen to map to some of the bacteria.

So worst case it looks to me like <<1%. The previous posts report 0.2% using amplicons, so I did another test.

Run A was a full MiSeq run of an amplicon library, the following run Run B was again really diverse, lots of different stuff.

If I search the undetermined bin of Run B for the first 15 bp of the amplicon, it's present 2497 times out of 12,746,730 reads, so again about 0.02%. Orthogonally, searching for the amplicon's exact barcode gives 925 or about 0.01%.

Searching for those 15 bp start seqs of that amplicon in all other samples in that run yields:
9 sequences out of 1,200,789 of a small RNA lib set (0.0007%)
10 sequences out of 3,193,181 of a bacterial RNA-seq set (0.0003%)

Something we can keep an eye on... hopefully no one every makes a decision based on a small number of reads! I've always taught that's a bad idea.

For the record - we do 0.5% tween maint. washes between all runs.
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Old 04-22-2013, 09:01 AM   #27
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It seems to me there are two predominant sources of error.

1) Library preparation (barcode reagents are cross-contaminated or PCR jumping, etc).

2) A platform specific source of cross-contamination from previous sequencing runs on the Miseq?

The first source I am quite familiar with, but the posts on this thread are indicating that somehow the previous library library templates are making their way into subsequent sequencing reactions (i.e. the second source)? How? Other than the library preparation being contaminated by templates from a previous library preparation, or non-aerosol resistant tips being used, or someone is not swapping tips during serial dilutions? I'm a bit confused on this.

-Tom
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Old 04-22-2013, 09:11 AM   #28
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Quote:
Originally Posted by thomasblomquist View Post
It seems to me there are two predominant sources of error.

1) Library preparation (barcode reagents are cross-contaminated or PCR jumping, etc).

2) A platform specific source of cross-contamination from previous sequencing runs on the Miseq?

The first source I am quite familiar with, but the posts on this thread are indicating that somehow the previous library library templates are making their way into subsequent sequencing reactions (i.e. the second source)? How? Other than the library preparation being contaminated by templates from a previous library preparation, or non-aerosol resistant tips being used, or someone is not swapping tips during serial dilutions? I'm a bit confused on this.

-Tom
Hi Tom

We can say with certainty that we're getting cross-contamination from previous sequencing runs.

Barcode contamination would give reads on the sequences represented within a run, but having unused barcodes (we see that, but at low representation). Cross contamination from previous runs gives reads that should not be represented in the current run, but identical to reads in the previous run (right down to the barcodes used in that run).

As far as I can tell (and validated by discussions with Illumina), this issue is caused by incomplete washing of the MiSeq plumbing between runs - small amounts of DNA probably adsorb to the tubing etc., and then are released while setting up the next run, where they hybridize to the flow cell and are sequenced.

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Old 04-22-2013, 09:32 AM   #29
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Quote:
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Hi Tom

We can say with certainty that we're getting cross-contamination from previous sequencing runs.

Barcode contamination would give reads on the sequences represented within a run, but having unused barcodes (we see that, but at low representation). Cross contamination from previous runs gives reads that should not be represented in the current run, but identical to reads in the previous run (right down to the barcodes used in that run).

As far as I can tell (and validated by discussions with Illumina), this issue is caused by incomplete washing of the MiSeq plumbing between runs - small amounts of DNA probably adsorb to the tubing etc., and then are released while setting up the next run, where they hybridize to the flow cell and are sequenced.

Cheers
h
Hmmm... Why would there be a complete "circuit" where reagents that go into the flow cell are recycled back later? Seems disasterous. Disclaimer: I only prep the libraries in house and send off to genomic cores for sequencing.

-Tom
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Old 04-22-2013, 09:46 AM   #30
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Though this may not be practical for all labs (who probably have access to a single MiSeq) as James pointed out in post #20 creative use of rotating barcodes should minimize this problem to a large extent (since the contaminants would end up in "undetermined" pile and be eliminated automatically).

It appears that we have a consensus that this problem exists. It is not going to be eliminated without specific protocol changes (if feasible) to ensure a crossover proof washing procedure or a hardware redesign (of parts of) MiSeq. I hope a path will exist to retro-fit the solution to existing machines if it has to be the latter.

There may be some applications (a small subset) where this contamination may be unacceptable and for those MiSeq may have to be passed over. But for all remaining applications this may become just another item in technology limitations/footnotes.
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Old 04-22-2013, 10:47 AM   #31
james hadfield
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Tom - the MiSeq moves your sample from the cartridge to teh flowcell through non-disposable tubing, there is a risk some carry-over will happen but this can be mitigated against with several relatively small changes.

Many applications will be largely unaffected, amplicons in heterogenous samples are likely to suffer the most.
Send your samples with different barcodes each time if you can.
Make sure to ask your facility to perform the washes between runs.

There is a hack to fool the MiSeq into thinking it is clean, don't use it! A maintensance wash is a bit of a pain but we'll be running them until this is fixed.
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Old 04-22-2013, 11:10 AM   #32
thomasblomquist
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Quote:
Originally Posted by james hadfield View Post
Tom - the MiSeq moves your sample from the cartridge to teh flowcell through non-disposable tubing, there is a risk some carry-over will happen but this can be mitigated against with several relatively small changes.

Many applications will be largely unaffected, amplicons in heterogenous samples are likely to suffer the most.
Send your samples with different barcodes each time if you can.
Make sure to ask your facility to perform the washes between runs.

There is a hack to fool the MiSeq into thinking it is clean, don't use it! A maintensance wash is a bit of a pain but we'll be running them until this is fixed.
Okay, that makes sense as to the source of the inter-run cross-contamination. Does the hi-seq operate in the same fashion?

Still, that seems like a bit of a design flaw. Why would an engineer design a non-disposable piece of tube for the loading on the chip? For that reason alone I would be hesitent to use this in the clinic.

-Tom
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Old 04-24-2013, 05:55 AM   #33
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Man, I need to check these forums more regularly. We've been seeing this problem in all of our amplicon runs. At first we assumed it was contamination during the sample prep, but after running two completely distinct amplicon types (16S and non-ribosomal gene amplicons made using very different fusion primers) we still found low level mixing of the two despite the samples never coming anywhere near each other except that they were both run on the same MiSeq.

I know my FAS said that the MiSeq tubing can't stand repeated exposures to KOH, so I guess that's out of the question as a cleaning solution, but hopefully they'll come up with something.
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Old 04-24-2013, 06:06 AM   #34
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Just how non-disposable is this tubing? I'd be willing to pay an extra $100 a run just to have contamination free assurance.

Can this topic be forwarded to someone at Illumina that is responsive to this need?

-Tom
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Old 04-24-2013, 06:11 AM   #35
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Originally Posted by thomasblomquist View Post
Just how non-disposable is this tubing? I'd be willing to pay an extra $100 a run just to have contamination free assurance.

Can this topic be forwarded to someone at Illumina that is responsive to this need?

-Tom
I'd say that the tubing is very non-disposable because from what my FAS and one of Illumina's field technicians have told me, in order to replumb the MiSeq you're looking at taking the whole system apart, including the optics, and would then have to put it all back together and re-align the optical table. My FAS said he once nicked one of the lines while doing an optical adjustment, and it took him an entire day to get just the one line replaced (I don't know which it was) and put the optics back in alignment.

Basically, Illumina will need to put out a new recommendation for a solvent that won't destroy the lines but will also sufficiently clean them to fix this issue.
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Old 04-24-2013, 06:12 AM   #36
thomasblomquist
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And the hiseq?

-Tom
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Old 04-24-2013, 06:41 AM   #37
james hadfield
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The tubing inside the MiSeq goes from the cartridge via a fixed sipper, through tubing to a vici valve, and through a final tube to the flowcell. It does not appear to be easily replaceable.

However a better wash is likely to decrease the carry-over.
Washing more frequently may well help.
Changing barcodes between runs should help.

These are all things we are in control of.

I think HiSeq 2500 comes with the option of cBot clustering if this is the case then there is almost no issue for 2500.
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Old 04-24-2013, 12:40 PM   #38
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Hi everyone, this is Kameran and I’m part of the Illumina Technical Support team. Illumina has posted a bulletin, “Best Practices for High Sensitivity Applications: Minimizing Sample Carryover”, which can be found by following this link:
https://icom.illumina.com/MyIllumina...applications-m


In summary, run to run sample carryover is more likely to have an effect on very low detection threshold applications. Our internal testing found that, when the instrument is properly washed and maintained, sample carryover typically remained below 0.1%, representing 1 read in 1000. Carryover may arise from a number of steps in the sequencing workflow, including incomplete removal of template DNA before the next run and various steps of library preparation. Proper maintenance procedures with water and Tween are recommended to minimize carryover. We currently do not advise the use of other wash additives, such as bleach, Triton or other decontamination solutions.

Of course, we’re always available by phone or email ([email protected]) to discuss any additional questions or concerns.
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Old 04-24-2013, 01:11 PM   #39
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Quote:
Originally Posted by kameran View Post
Hi everyone, this is Kameran and I’m part of the Illumina Technical Support team. Illumina has posted a bulletin, “Best Practices for High Sensitivity Applications: Minimizing Sample Carryover”, which can be found by following this link:
https://icom.illumina.com/MyIllumina...applications-m


In summary, run to run sample carryover is more likely to have an effect on very low detection threshold applications. Our internal testing found that, when the instrument is properly washed and maintained, sample carryover typically remained below 0.1%, representing 1 read in 1000. Carryover may arise from a number of steps in the sequencing workflow, including incomplete removal of template DNA before the next run and various steps of library preparation. Proper maintenance procedures with water and Tween are recommended to minimize carryover. We currently do not advise the use of other wash additives, such as bleach, Triton or other decontamination solutions.

Of course, we’re always available by phone or email ([email protected]) to discuss any additional questions or concerns.
Thank you Kameran. What is the rate of decrease over multiple uses and washes? Specifically, using a shared instrument, how many other samples should I interdigitate between returning to previously used barcode combinations to achieve a 1:10,000; 1: 100,000, etc. carry-over?

-Tom
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Old 04-25-2013, 03:24 PM   #40
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Hi Tom,



Studies regarding barcode integrations and degradation rate for run to run carryover have not been performed. However, customers have reported steady decreases from wash to wash. Anecdotal evidence suggests that, if carryover was 1 in 1000, it would be more like 1 in 1000000 the next time. This assumes there isn’t template that has dried and gotten “stuck” somewhere in the system. The more rinsing we can do on the MiSeq, the lower the carryover rate is expected to be.
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