MiSeq Carry-over contamination between runs

[Harlon]

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

[kcchan]

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?

[bbeitzel]

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.

[Bucky]

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.

[pmiguel]

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).

--
Phillip

[ECO]

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.

[bbeitzel]

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).

[pmiguel]

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?

--
Phillip

[pmiguel]

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.

--
Phillip

[bbeitzel]

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.

--
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.

[Harlon]

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

[GW_OK]

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.

[GW_OK]

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

[thomasblomquist]

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/technical-vault/decoded-archive/competitive-edge/decoded/2012/09/21/reduced-barcode-contamination-using-oligonucleotides-with-trugrade-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

[james hadfield]

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.