Thursday, 14 August 2014

How many MiSeq owners are using the bleach protocol to minimise carryover?

A comment popped up on a post I'd written in April last year "MiSeq (and 2500) owners better read this and beware"that made me think I'd ask readers the question in the title: "how many of you are using the MiSeq bleach wash protocol?

The carryover issue led to a small residue of the last library to be run being sequenced in the subsequent run. This caused a potential problem to MiSeq users, particularly those interested in low frequency mutations. My post was prompted after some discussions with other MiSeq owners and a thread on SEQanswers, which Illumina posted to describing their experiences with reducing this carryover, and that it was seen typically at 0.1%.

The comment on my post was about a more aggressive bleach protocol which reduced carryover to almost undetectable levels (thanks Illumina), but that appears to have not been communicated to all users. It was impossible for me to find on the Illumina website but it's not the most easy to navigate site in the world so I thought I'd put the document up here for you to see (click the image for the TechNote).

https://drive.google.com/file/d/0B383TG7oJh2CTWp6MzZkMHBDd28/edit?usp=sharing

You need to request this through your techsupport team as it needs a new recipie on your MiSeq. And you really must follow the instructions to the letter, too much bleach and you'll probably kill your MiSeq!

Ilumina demonstrated that this protocol can reduce carry-over to less than 0.001% or one read per 100,000. We've been using this as the default wash for many months and reports of carry-over are nearly non-existent.

6 comments:

  1. I think you should point out that the NextSeq does this by default!

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  2. Yes that's right, the NextSeq has very nicely been engineered to complete the wash protocol as part of the run. I did not know it also incorporated a bleach wash. Well done NextSeq team!

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  3. That's what happens when you carve the innards of your machine out of a solid block of resin!

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  4. The nextseq carryover is a huge problem for us now.

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    1. We are having massive problems with run to run carryover on the NextSeq but are being told it has not been reported previously. Have you got anywhere with your carryover problem?

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    2. We use the bleach protocol every time and see very little carryover. I thought the NextSeq cartridge had a bleach wash built in? If you've ruled out contamination of the libraries upstream then I'd push your FAS to escalate the issue, especially if you can see your last run in a PhiX control run!

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