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Old 09-16-2013, 08:07 AM   #1
bbm
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Default which kit to use for whole genome BS-seq library?

Hi all,

I started a BS-seq whole genome project to look at nutrition effect on insect geome. I try to decide which kit to use for making the whole genome DNA library. Now it down to 3 candidates.

1) Illumina/epicenter EpiGnome™ Methyl-Seq Kit for 12 reactions. $95/reaction


2) Nugen Ovation® Ultralow Methyl-Seq Library Systems for 32 reactions, $47/reaction

or 3) Illumina TruSeq DNA sample prep kit for 48 reactions, $49/raction

Two other experts I have talked to used #3, but they are in big sequencing center, and this product is going to be discontinue after Dec. this year. Illumina promotes the #1 but I don't know if that method has any bias or not because of ssDNA.

I'm in a start up lab, and budget is tight...I have total 6 libraries to make, and also 2-3 to practice before I start the real sample. I know I need buy Zymo kit for BS-trt, and Kappa kit for better PCR.

I would appreciate your opinion or advice on which kit to use?!

Last edited by bbm; 09-16-2013 at 08:09 AM.
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Old 09-20-2013, 12:04 PM   #2
bbm
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no comments? anybody?
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Old 09-20-2013, 01:20 PM   #3
kwaraska
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New England BioLabs has good kits, small sizes and I don't think you need a methyl-seq kit but you might need a microbiome kit.

I would look into them. Their kits are Illumina sequencer compatible
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Old 09-23-2013, 11:54 AM   #4
Olaf Blue
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What is your input? We have used Epicentre’s methyl-seq kit and it works very well with low input amounts, and the libraries we have sequenced so far have much higher diversity compared to other kits. Pre-library BS-conversion has many advantages as well. My recommendation would be to go with the EpiGnome kit from Epicentre.
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Old 09-23-2013, 07:58 PM   #5
bbm
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Quote:
Originally Posted by Olaf Blue View Post
What is your input? We have used Epicentre’s methyl-seq kit and it works very well with low input amounts, and the libraries we have sequenced so far have much higher diversity compared to other kits. Pre-library BS-conversion has many advantages as well. My recommendation would be to go with the EpiGnome kit from Epicentre.
My input is small tissue from aphids. Is there any bias with ssDNA pre-library BS-conversion? Thanks a lot!
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Old 09-24-2013, 06:26 AM   #6
Olaf Blue
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Hi, the EpiGnome product uses the Zymo Gold or Zymo Lightning conversion kits; Zymo claims no bias and can be used with as little as 500 pg input genomic DNA. The DNA post-conversion is used to construct the libraries for sequencing on Illumina paired end flow cells. The PCR primers used for library amplification/enrichment are not methylated; methyalted primers could be used but from what we understand they're insanely expensive.
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Old 09-25-2013, 09:18 AM   #7
bbm
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Quote:
Originally Posted by Olaf Blue View Post
Hi, the EpiGnome product uses the Zymo Gold or Zymo Lightning conversion kits; Zymo claims no bias and can be used with as little as 500 pg input genomic DNA. The DNA post-conversion is used to construct the libraries for sequencing on Illumina paired end flow cells. The PCR primers used for library amplification/enrichment are not methylated; methyalted primers could be used but from what we understand they're insanely expensive.
Hi Olaf Blue,
Thank you for helping me and all the infor. I think I'll try the epicentre kit. What software or tool do you use for sequence data analysis? Is the BS reads in one of the 4 forms (+fw, +rc, -fw, -rc) or just 2 forms ? Thanks again!
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Old 09-25-2013, 10:52 AM   #8
Olaf Blue
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Hi BBM, basically the way we do the data analysis is (1) to trim off the first 6 bases off of the 5'-end, followed by adapter trimming using "Trimomatic" (I am not a Bioinformatcian so I get this information from my Core facility). After adper sequences are trimmed, then the sequence analysis is performed using Bismark. If you have anything more specific, let me know.

Olaf
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Old 09-25-2013, 01:42 PM   #9
anth
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Quote:
Originally Posted by Olaf Blue View Post
Hi BBM, basically the way we do the data analysis is (1) to trim off the first 6 bases off of the 5'-end, followed by adapter trimming using "Trimomatic" (I am not a Bioinformatcian so I get this information from my Core facility). After adper sequences are trimmed, then the sequence analysis is performed using Bismark. If you have anything more specific, let me know.

Olaf
Hello Olaf,

Sorry to thread hijack. I'm very curious as to how you approach analysis. Following the methylation calls from Bismark, how do you identify differential methylation?

Thanks!

Anthony
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Old 10-07-2013, 10:13 PM   #10
bbm
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Quote:
Originally Posted by Olaf Blue View Post
Hi BBM, basically the way we do the data analysis is (1) to trim off the first 6 bases off of the 5'-end, followed by adapter trimming using "Trimomatic" (I am not a Bioinformatcian so I get this information from my Core facility). After adper sequences are trimmed, then the sequence analysis is performed using Bismark. If you have anything more specific, let me know.

Olaf
Hi Olaf,
Does the strandness matter? I assume the epiGnome kit doesn't produce the stranded library. just curious... BTW: I got the epiGnome kit and testing it out... Do you measure the yield after BS conversion? Did you use 50 ng as the epignome protocol suggested before BS conversion?
Thanks.

Regards,
BBM
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Old 10-07-2013, 10:14 PM   #11
bbm
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Quote:
Originally Posted by Olaf Blue View Post
Hi BBM, basically the way we do the data analysis is (1) to trim off the first 6 bases off of the 5'-end, followed by adapter trimming using "Trimomatic" (I am not a Bioinformatcian so I get this information from my Core facility). After adper sequences are trimmed, then the sequence analysis is performed using Bismark. If you have anything more specific, let me know.

Olaf
Thank you very much for your input!

Regards,
Hongmei
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Old 10-10-2013, 08:08 AM   #12
james hadfield
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Why not look at Cambridge Epigenetix's new True Methylome for oxidative bisulfite sequencing (oxBS-Seq)?
http://www.cambridge-epigenetix.com/...1/truemethyl-6
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Old 11-06-2013, 02:43 PM   #13
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Hi,

I have recently used a mix of kits successfully: Qiagen EpiTect Bisulphite Kit followed by EpiGnome Methyl-Seq kit and then sequenced on MiSeq. All very easy and straightforward.

Hope that helps,
Justin
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Old 11-21-2013, 02:20 PM   #14
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Justin,

Would you be able to give more details about how this combination of kits worked for you?

In particular:
- What was your input DNA?
- Did you fragment your DNA in any way before BS-conversion?
- What was the size range of your fragments after BS-conversion and after library prep and sequencing?

Thanks!
Evelien
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Old 11-25-2013, 01:44 PM   #15
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Hi Evelien,

The combination was quite straight forward. My input DNA was from plant leaf tissue freshly extracted using a Qiagen DNeasy kit (final elution in DNase/RNase free ultra pure water). Then simply follow the manuals as-is. No specific fragmentation is needed as the bisulphite treatment does this anyway. The range of fragments post treatment was between 150 and 2000 bp (peak around 300 bp) measured on a bioanalyzer high sensitivity chip.

Hope that helps,

Justin
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