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smice 05-26-2016 03:51 AM

Experienced sequencing providers for RRBS-seq?

can someone recommend me a reliable sequencing provider for RRBS-sequencing? Our current provider struggles with this kind of application, they have problems finding the right amount of PhiX, they generate low quality bases and waste a lot of reads; they have admitted that this is the first time they try their hands at this application, and so they do not have the expertise with sequencing such low-complexity libraries.

We have a lot of urgent RRBS-seq projects in the pipeline, and we would like to do RRBS-seq regularly in the future, so we are considering changing the sequencing provider.

So I'm asking mainly the RRBS users (but WGBS users are also welcome of course, though I think the complexity is better with WGBS): where do you usually sequence, which sequencing provider can you recommend that has experience with sequencing low complexity libraries?

Also we prefer Illumina technology and situation in Europe, but these are not priorities.

Thanks in advance!

nucacidhunter 05-26-2016 04:19 AM

I cannot recommend any sequence provider but there is a RRBS kit which prepares high diversity RRBS libraries. The libraries can be sequenced without or low PhiX spike-in (1%) with clustering 10% below shotgun libraries.

smice 05-28-2016 11:05 AM

Thanks for the reply! And which kit would this be? Have you tried it in practice? Do you know maybe how do they achieve this?

Nevertheless we still need someone to sequence it, so if anyone has any suggestions for a NGS service provider, don't hold it back. :)

nucacidhunter 05-28-2016 04:35 PM

You can have a look for posts #9 onwards in the following thread:

DNATECH 05-30-2016 12:30 PM

Sequencing RRBS libraries (standard protocol) on the HiSeq 4000 has not been a problem for us - one has to dose in 30 to 40% of a balanced library though

smice 06-02-2016 02:31 AM

Thank you for the answers!

AllSeq 06-02-2016 10:45 AM

We've found that RRBS is an application that a lot of providers are simply not comfortable running. While it's not a direct answer to your question, I'd encourage you to try out AllSeq's Sequencing Marketplace to see if you can find any who are interested. It's completely free to use for both buyers AND providers (so no hidden fees driving up your project costs). Best of luck!

clolalan7 10-13-2016 04:17 PM

Do you use a specific type of balanced library along with your RRBS ones? I would imagine that a library with the same range of amplicon sizes as your RRBS' would be necessary?

Thanks in advance!

DNATECH 10-13-2016 05:02 PM

Hi clolalan,

yes, somewhat similar fragment lengths make the dosing much easier. RNA-seq libraries tend to be perfect.

clolalan7 10-14-2016 04:52 AM

Thank you DNATECH,

I will suggest this to my colleagues

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