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Old 06-05-2014, 02:10 PM   #1
lyndaben
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Default which kit for RRBS?

Any advice on which kit to use for RRBS? I'm looking at Epignome Methyl-Seq; NuGen Ovation Ultra low Methyl-seq; Bioo NEXTflex bisulfite sequencing kit,
The Illumina web site is confusing - their RRBS workflow includes ligation of methylated adapters which are no longer available. I called to ask about the Epignome kit for RRBS and was advised not to do RRBS at all and use microarrays (or WGBS - which is cost prohibitive for a new lab start-up). Any advice for a newbie to RRBS would be much appreciated

Ultimately I want to use FFPE samples - impossible?
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Old 07-24-2014, 08:05 AM   #2
amdic2
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Default Which kit for RRBS

Dear lyndaben,
Have you decided which kit you are going to use?
I am also currently looking at several options.
The protocol published by Boyle et al. (2012) Genome Biology appears quite easy to optimize. I am also interested by the NextFlex kit.
By the way, Boyle et al. mentionned to used the bisulfite conversion protocol for FFPE samples with ANY samples. It thus doesn't appear to be a problem?
Cheers,
Anne-Marie
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Old 07-24-2014, 08:09 AM   #3
lyndaben
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I opted for the NuGEN Ovation kit. They provided me with a protocol addition for RRBS. It was just delivered this week so I have not had the chance to try it yet
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Old 03-04-2015, 09:41 AM   #4
MasterSeq
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I know this is an old thread but this could be useful for somebody: NuGEN just launched a kit specifically for RRBS which should be much better than using their Methyl-Seq kit. It uses direct ligation from MspI digestion, only one bead purification and no need for phiX spike-in to deal with the lack of diversity coming from the MspI ends.
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Old 03-13-2015, 06:59 AM   #5
lyndaben
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The new kit will be better hopefully. A note of caution to anyone using the old kit with the modified RRBS protocol: The first sequencing run we multiplexed with RNAseq samples and everything was great and the data looks good. This last time we multiplexed with PhiX (in 6 of 8 lanes) as recommended by NuGEN and we are having problems with the data (only 3-10M reads for the RRBS samples). On the same flow cell, two of the lanes were multiplexed with RNAseq and we got 30M reads for the RRBS samples, so we know it was not a sequencing problem or a library problem.
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Old 03-20-2015, 03:27 AM   #6
chapmandu2
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Out of interest, has anyone tried the Methyl-Seq hybrid capture sequencing method from Agilent instead of RRBS?
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Old 03-20-2015, 11:40 PM   #7
nucacidhunter
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This kit does not seem to be popular. I could not find any publication that have used it nor Agilent Techsupport was able to provide any reference. It would cost more than RRBS for the same coverage of target region, noting that target or covered regions are different between two methods.
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Old 03-24-2015, 07:23 PM   #8
Genohub
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Default Methyl-DNA Sequencing

We've posted several different flavors of Methyl-DNA sequencing. Use the links at the end of the post to see the kits that NGS service providers on Genohub use.

- Genohub
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Old 05-15-2015, 11:40 AM   #9
NuGEN
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Default Dedicated kit for RRBS from NuGEN

I wanted to comment that NuGEN has recently launched a dedicated kit for RRBS, the Ovation RRBS Methyl-Seq System - details can found here

http://www.nugen.com/nugen/index.cfm...yl-seq-system/
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Old 09-29-2015, 05:57 AM   #10
Faria
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Quote:
Originally Posted by lyndaben View Post
I opted for the NuGEN Ovation kit. They provided me with a protocol addition for RRBS. It was just delivered this week so I have not had the chance to try it yet
Dear lyndaben,

How did your data turn out? Did you have any issues demultiplexing your samples? We recently used the kit. The libraries looked great but we are having issues with demultiplexing. Any advice/ tricks?

Thanks for your time
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Old 09-29-2015, 06:32 AM   #11
lyndaben
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We used the NuGEN Ovation kit with great results when we multiplexed our samples with RNA-seq samples. When we multiplexed some RRBS libraries with PhiX we had problems with demultiplexing but when we re-sequenced the same samples and multiplexed with RNA-seq libraries we had no problems at all.

We are now making libraries from their new Ovation RRBS Methyl-seq kit. You do not need to multiplex with high quantities of complex libraries with this kit. Library construction is working great but we haven't sequenced any yet.

BTW, we had the same demultiplexing problem when we had some TrueMethyl-seq RRBS and oxRRBS libraries sequenced with PhiX and again no problem when the same libraries were re-sequenced with some RNA-seq libraries
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Old 09-29-2015, 08:37 AM   #12
nucacidhunter
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Demultiplexing rate with new kit is low with or without PhiX. Undetermined folder reads have index but it seems to have various sequences. NuGEN should have seen this issue if they sequenced libraries according to their recommendation.
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Old 09-29-2015, 08:56 AM   #13
Faria
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Would you mind sharing your % undertermined reads?
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Old 09-29-2015, 09:00 AM   #14
nucacidhunter
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15-30% depending on lane. Multiplexing level does not seem to have any effect.
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Old 09-29-2015, 09:00 AM   #15
lyndaben
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Faria, did you use the old kit or the new kit?
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Old 09-29-2015, 09:06 AM   #16
Faria
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We used the new kit but our bioinformatician has been unsuccessful with the demux using Nugen's protocol. We are trying to figure out the source of the problem.
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Old 09-29-2015, 09:10 AM   #17
lyndaben
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If you figure it out, please would you post the solution? We are prepping libraries with the new kit at the moment, so it would be good to know before we have them sequenced
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Old 09-29-2015, 09:12 AM   #18
Faria
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Will do! Please keep me posted on how yours turn out.
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Old 09-29-2015, 05:14 PM   #19
luc
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Are the indices by chance not properly methylated or do they seem contaminated with unrelated sequences?

Quote:
Originally Posted by nucacidhunter View Post
Demultiplexing rate with new kit is low with or without PhiX. Undetermined folder reads have index but it seems to have various sequences. NuGEN should have seen this issue if they sequenced libraries according to their recommendation.
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Old 09-29-2015, 05:52 PM   #20
nucacidhunter
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Sequences of part of NuGEN P5 adapter is not in public domain (they use custom read 1 sequencing primer). I think they ligate partial adapters (methylated) and then add index and restore full adapter sequence after conversion with PCR. Undetermined reads’ index seems to be random indicating either sequencing or synthesis error. Sequencing error and sequencing primer synthesis issues can be excluded because other lanes in flow cell are demultiplexing with higher rate (95-99%). That narrows down the possible cause to adapter or PCR primer synthesis error.

They are not from contamination because reads have very low C and high T content indicating conversion and map to reference.

Edit: Further analysis indicates that they use Y adapters in which P5 sequences are non-methylated, but P7 sequences are.

Last edited by nucacidhunter; 10-25-2015 at 08:54 PM. Reason: Extera info added.
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