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Old 12-03-2015, 12:35 AM   #1
Andersen
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Location: Copenhagen

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Default Multiplexing RRBS samples

Dear everyone,

Im preparing a RRBS library. I want to multiplex 10 samples from 150ng DNA.

We use ilumnia DNA nano LT adaptors on the fragmented DNA.

I have been looking online for multiplexing guidelines, but cannot seem to find anything on what is recommended.

I used these adaptors:
TRUSEQ_1 ATCACG
TRUSEQ_2 CGATGT
TRUSEQ_4 TGACCA
TRUSEQ_5 ACAGTG
TRUSEQ_6 GCCAAT
TRUSEQ_7 CAGATC
TRUSEQ_8 ACTTGA
TRUSEQ_10 TAGCTT
TRUSEQ_11 GGCTAC
TRUSEQ_12 CTTGTA

Would this multiplexing with the converted DNA cause too low diversity of clusters or could it be manageable?

We would spike in with 30% PhiX as allways to reduce cluster diversity.

We use a protocol similar to this:
http://www.genomebiology.com/2012/13/10/R92

Thank you for your help.
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Old 12-03-2015, 01:21 AM   #2
nucacidhunter
Jafar Jabbari
 
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For multiplex sequencing of more than six Nano LT indexes, you do not have to be concerned with index diversity so your ten-plex will be fine. If you want detailed compatibility tables you can find them in TruSeq Nano library prep user guide. Nano adapters have mC so they do not get converted by bisulfite. Obviously, you will need more than one sequencing lane if they are mouse libraries.

There is a new commercial kit which directly ligates adapters to MspI overhang and includes 0-3 diversity bases. These libraries do not require PhiX spike-in and can be sequenced like standard libraries with 10-20% less cluster.
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Old 12-03-2015, 01:29 AM   #3
Andersen
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Location: Copenhagen

Join Date: Oct 2015
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Quote:
Originally Posted by nucacidhunter View Post
For multiplex sequencing of more than six Nano LT indexes, you do not have to be concerned with index diversity so your ten-plex will be fine. If you want detailed compatibility tables you can find them in TruSeq Nano library prep user guide. Nano adapters have mC so they do not get converted by bisulfite. Obviously, you will need more than one sequencing lane if they are mouse libraries.

There is a new commercial kit which directly ligates adapters to MspI overhang and includes 0-3 diversity bases. These libraries do not require PhiX spike-in and can be sequenced like standard libraries with 10-20% less cluster.
Wow, thank you very much for your quick and precise answer.

Sounds really nice, do you have the name of one of these suppliers?
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Old 12-03-2015, 01:33 AM   #4
nucacidhunter
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NuGEN Ovation RRBS kit. There is a bit of discussion regarding the kit in this thread: http://seqanswers.com/forums/showthr...rbs#post181746
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library generation, library pooling, methylation, multiplex illumina pool, rrbs

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