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Old 04-08-2013, 05:02 PM   #1
Harlon
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Default MiSeq Carry-over contamination between runs

Hi All

We appear to be suffering from carry over contamination in our MiSeq runs - i.e. if we sequence a DNA sample in one MiSeq run, we see about the same sample in the subsequent run.

Measured in terms of reads, we see about 0.2% contamination run-to-run - i.e. if we see 10,000 reads of a given amplicon/barcode in one run, we'll see ~20 reads in the following run, even if that amplicon/barcode pair was absent from the prep.

Important notes about our workflow:
- Barcodes are added by PCR (we are using our own library prep, not Nextera, etc).
- We perform a post-run wash and a maintenance wash after every run.

I am quite certain that this is carry-over within the MiSeq, and that it is actually carry-over, and not simply barcode contamination within the primers. On the first run of an amplicon, it only shows up with its assigned barcode. It is also detected in the subsequent run. I'm quite certain this is also not laboratory contamination.

After speaking with Illumina, this is certainly feasible - they are aware of this issue, although they do see less run-to-run contamination than we see. They suggested that we do 2 maintenance washes between runs, which seems like a lot, and that we don't pour bleach into it, which was certainly not my plan.

Has anyone else had similar issues, and more importantly, does anyone know of any solutions?

Thanks!
Harlon
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Old 04-08-2013, 08:23 PM   #2
kcchan
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I would imagine that the carryover issue is caused by the sipper in position 17, the template well. Perhaps you can run a few extra washes only at that position if you don't want to do another maintenance wash?

One other thing you should be doing is changing out the water/tween solution with every wash cycle, but I would hope that you're already doing that.

It may also be possible that the contamination is occurring on your bench. A contaminated tube of primers or buffer could be a culprit. Have you tried cleaning your workbench and using all new reagents in your second library prep?
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Old 04-09-2013, 04:47 AM   #3
bbeitzel
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We see the carryover as well at around the same % you are seeing it. We just updated to the most recent RTA/MCS, and in the new manual they change the wash to 0.5% Tween instead of water. We'll see if that reduces / removes the carryover.
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Old 04-09-2013, 06:25 AM   #4
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Same thing here, 0.5% Tween doesn't seem to help much. We started using different bar codes for each run. We generally see about 0.1% carry over, even after performing an extra post-run wash.
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Old 04-09-2013, 07:00 AM   #5
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Is everyone changing the 0.5% Tween in the water tray between each of the 3 stages of the maintenance wash?

Does anyone have 2 MiSeqs that they could use to verify that the contamination does not derive from amplicon contamination during library construction/amplification? Or, alternatively, two independent sources of libraries (from labs that share no space, reagents, equipment or staff).

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Old 04-09-2013, 09:31 AM   #6
ECO
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This will likely be a problem on the HiSeq2500 as well (same sippers and lines, etc)...I don't have enough data to say anything definitive yet.
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Old 04-09-2013, 12:34 PM   #7
bbeitzel
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Quote:
Originally Posted by pmiguel View Post

Does anyone have 2 MiSeqs that they could use to verify that the contamination does not derive from amplicon contamination during library construction/amplification? Or, alternatively, two independent sources of libraries (from labs that share no space, reagents, equipment or staff).

--
Phillip
We ran some libraries for another group that they had prepared in a totally separate lab. They picked up reads from our previous run in their data, and we picked up reads from their libraries in our subsequent run. The subsequent run was after a maintenance wash (3X, water only at that point).
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Old 04-09-2013, 12:48 PM   #8
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Quote:
Originally Posted by ECO View Post
This will likely be a problem on the HiSeq2500 as well (same sippers and lines, etc)...I don't have enough data to say anything definitive yet.
The NaOH wash used for the HiSeq2500 may be more effective than the 0.5% Triton used for the MiSeq. Or not.

Hmm, so the cBot has single-use manifolds. But there is tubing inside the instrument itself. Anyone have figures for amount of bleed over run to run on the cBot?

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Old 04-09-2013, 12:51 PM   #9
pmiguel
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Quote:
Originally Posted by bbeitzel View Post
We ran some libraries for another group that they had prepared in a totally separate lab. They picked up reads from our previous run in their data, and we picked up reads from their libraries in our subsequent run. The subsequent run was after a maintenance wash (3X, water only at that point).
Hmm, not good. TritonX should help though. Do you wait long after a run to do the post-run wash? Might be easier to wash off amplicons if they don't get a chance to dry.

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Old 04-09-2013, 12:57 PM   #10
bbeitzel
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Quote:
Originally Posted by pmiguel View Post
Do you wait long after a run to do the post-run wash? Might be easier to wash off amplicons if they don't get a chance to dry.

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Phillip
We usually do the post-run wash as soon as the run finishes. If it finishes overnight, it gets washed first thing the next morning.
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Old 04-10-2013, 08:23 AM   #11
Harlon
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We've always run 0.5% Tween in our maintenance washes, and we do switch buffers in both the bottle and the tray in between each step of the wash. We also almost always do our post-run wash within a couple of hours of the end of the run.

As far as NaOH goes, when we contacted Illumina, they stated NaOH is very difficult to rinse out of the machine, and that it tends to cause more problems than it solves. I'm pretty reluctant to go dumping any caustics into the instrument - my gut tells me that if you dilute down to the point where they aren't dangerous to the machine, they probably won't be too effective at removing DNA.

Triton-X sounds interesting, however. Does anyone know how Triton-X and Tween compare in reducing background?

Thanks for all the help!
h
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Old 04-10-2013, 09:29 AM   #12
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Quote:
Originally Posted by pmiguel View Post
The NaOH wash used for the HiSeq2500 may be more effective than the 0.5% Triton used for the MiSeq. Or not.

Hmm, so the cBot has single-use manifolds. But there is tubing inside the instrument itself. Anyone have figures for amount of bleed over run to run on the cBot?
Nothing can carry over in the cBot, everything that touches the DNA or the flowcells is single use only.
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Old 04-10-2013, 09:39 AM   #13
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I still have a big jug of Contrad-70 from the old Cluster Station/GA2x days. I've thought about using it instead of Tween. It's supposed to be extremely rinse-able and the manufacturer (DECON) claims it will wash away anything....

http://www.deconlabs.com/products.php?ID=2
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Old 04-11-2013, 09:49 AM   #14
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Barcode contamination from the synthesis and preparation steps is a HUGE issue. A lot of care for stringency in all steps must be taken. Don't use plates to prep sequencing libraries in parallel!!!

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245947/

Also,

http://www.idtdna.com/pages/support/...ade-processing

Be careful with barcoding and multiplexing. As with any experiment, include controls, etc. to monitor level of cross-contamination -talk between barcodes, etc.

-Tom
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Old 04-17-2013, 01:28 AM   #15
james hadfield
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We're going to take a look at previous MiSeq runs. I'd encourage anyone reading this to report back if you do the same.

We're also taking the simple stpe of making sure we don't run the same barcodes on the next run. This should reduce the problem back to almost background and is a quick fix. hopefully a robust wash protocol wil completely clear the issue.
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Old 04-17-2013, 05:00 AM   #16
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Just wanted to chime in and say that we've seen run-to-run contamination too on our MiSeq.

After reading this post, I looked into it and found plenty of evidence.

Sam
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Old 04-17-2013, 09:38 AM   #17
Harlon
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Hi All

I just read James' blog post on this:
http://core-genomics.blogspot.ca/201...read-this.html
I'm not certain that my original statement of the problem is entirely clear, and I want to make sure people don't get overly concerned based on our data.

What we're seeing is 0.2% of reads carrying through from run to run. This means that if we saw 100k reads on BRAF V600E last run, we'd see 200 reads on BRAF V600E in the current run.

My take is that the impact on sensitivity is read# based, not abundance based. This would affect sensitivity where:
1) The previous run had a very high mutation abundance, and similar total read number, or
2) The previous run had a moderate mutation abundance, but much higher read coverage.

In both cases, it should be possible to calculate bleed-through and account for the background in downstream analysis - by examining previous runs, we should be able to predict whether carry-over will be a problem with the current run. To take James' example, if read number on BRAF was identical between runs, previous run had 100% V600E, I'd expect to see 0.2% in the current run, and flag that as a problem.

In any case, we're planning to start running a lot more barcodes, so that we can rotate them between runs. I'm hoping our carry-over will decay to near zero by the time we re-run a barcode set. That said, I really think we'll need to track this carefully - one could imagine that carry-over decays slowly...
We won't have results on this for a while, but when we get them, we'll post them.

Cheers
harlon

Last edited by Harlon; 04-17-2013 at 09:39 AM. Reason: added weblink to James' blog post
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Old 04-17-2013, 10:47 AM   #18
thomasblomquist
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Quote:
Originally Posted by Harlon View Post
Hi All

I just read James' blog post on this:
http://core-genomics.blogspot.ca/201...read-this.html
I'm not certain that my original statement of the problem is entirely clear, and I want to make sure people don't get overly concerned based on our data.

What we're seeing is 0.2% of reads carrying through from run to run. This means that if we saw 100k reads on BRAF V600E last run, we'd see 200 reads on BRAF V600E in the current run.

My take is that the impact on sensitivity is read# based, not abundance based. This would affect sensitivity where:
1) The previous run had a very high mutation abundance, and similar total read number, or
2) The previous run had a moderate mutation abundance, but much higher read coverage.

In both cases, it should be possible to calculate bleed-through and account for the background in downstream analysis - by examining previous runs, we should be able to predict whether carry-over will be a problem with the current run. To take James' example, if read number on BRAF was identical between runs, previous run had 100% V600E, I'd expect to see 0.2% in the current run, and flag that as a problem.

In any case, we're planning to start running a lot more barcodes, so that we can rotate them between runs. I'm hoping our carry-over will decay to near zero by the time we re-run a barcode set. That said, I really think we'll need to track this carefully - one could imagine that carry-over decays slowly...
We won't have results on this for a while, but when we get them, we'll post them.

Cheers
harlon
Thank you for your response. Our lab has been running known negative and known positive controls, and for each patient sample performing two technical replicates by barcoding twice with two seperate barcode reagents. Results must be concordant between the two technical replicates, as well as 5 SD above our LOD in the negative control (i.e. analytical LOQ).

-Tom
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Old 04-17-2013, 05:40 PM   #19
vdauwera
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FYI the GATK callers now include a fractional downsampler specifically designed to compensate for contamination (regardless of origin, which can be lab prep, machine etc). It is activated by default for an estimated rate of contamination of 5% (iirc) but the rate can be adjusted if you suspect a larger issue. So for most genotyping purposes at least this doesn't have to be a showstopper.
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Old 04-17-2013, 11:45 PM   #20
james hadfield
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We have started to rotate the use of barcodes. We were already duplicating data sets (although not with sepearate barcodes). ANd we will probably perform a maintaenance wash between runs.
All of this will mean the problem only exists as an issue for those few groups looking for very low mutation rates in amplified samples (like some at CI). Those grous have lots of other issues to contend with as well so this is just one more to add to the mix.
I am sure we'll see an improved wash from Illumina soon and that should be the end of it for most users.
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