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-   -   which kit to use for whole genome BS-seq library? (http://seqanswers.com/forums/showthread.php?t=33754)

bbm 09-16-2013 09:07 AM

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?!

bbm 09-20-2013 01:04 PM

no comments? anybody?

kwaraska 09-20-2013 02:20 PM

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

Olaf Blue 09-23-2013 12:54 PM

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.

bbm 09-23-2013 08:58 PM

Quote:

Originally Posted by Olaf Blue (Post 116969)
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!

Olaf Blue 09-24-2013 07:26 AM

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.

bbm 09-25-2013 10:18 AM

Quote:

Originally Posted by Olaf Blue (Post 117067)
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!

Olaf Blue 09-25-2013 11:52 AM

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

anth 09-25-2013 02:42 PM

Quote:

Originally Posted by Olaf Blue (Post 117194)
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

bbm 10-07-2013 11:13 PM

Quote:

Originally Posted by Olaf Blue (Post 117194)
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

bbm 10-07-2013 11:14 PM

Quote:

Originally Posted by Olaf Blue (Post 117194)
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

james hadfield 10-10-2013 09:08 AM

Why not look at Cambridge Epigenetix's new True Methylome for oxidative bisulfite sequencing (oxBS-Seq)?
http://www.cambridge-epigenetix.com/...1/truemethyl-6

EpiBrass 11-06-2013 03:43 PM

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

Embunnik 11-21-2013 03:20 PM

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

EpiBrass 11-25-2013 02:44 PM

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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