Multiplexing RRBS samples

Andersen · 12-03-2015, 01:35 AM

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.

nucacidhunter · 12-03-2015, 02:21 AM

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.

Andersen · 12-03-2015, 02:29 AM

Quote:

Originally Posted by nucacidhunter

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?

nucacidhunter · 12-03-2015, 02:33 AM

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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12-03-2015, 01:35 AM	#1
Andersen Member Location: Copenhagen Join Date: Oct 2015 Posts: 15	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.

12-03-2015, 02:21 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	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.

12-03-2015, 02:33 AM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	NuGEN Ovation RRBS kit. There is a bit of discussion regarding the kit in this thread: http://seqanswers.com/forums/showthr...rbs#post181746