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consider gigas MBD bisulfite sequencing #275

Closed yaaminiv closed 5 years ago

sr320 commented 6 years ago

Awaiting DNA extractions - @yaaminiv can you get @kaitlynrm started on something related to this?

yaaminiv commented 6 years ago

@kaitlynrm Let's start extracting DNA Thursday morning! I can help when I'm not in class. I'll write up a protocol and post it here.

sr320 commented 6 years ago

please put protocol here: https://github.com/RobertsLab/resources/tree/master/protocols

and provide url here

kaitlynrm commented 6 years ago

Do you want me to pre-scrape any blocks @yaaminiv ?

yaaminiv commented 6 years ago

@kaitlynrm Not that I know of. If you and @kubu4 have time, could you take some of our old samples and run them on the Bioanalyzer? I'd like to know fragment lengths. The samples are in a box in the fridge and they have my name on them.

yaaminiv commented 6 years ago

Protocol can be found here. Lysis method may be modified when I look at Qubit results later today.

@kaitlynrm, you can find the plan for tomorrow in this lab notebook post.

kubu4 commented 6 years ago

Heads up, Step 11 (cleaning Tissue Tearer) is not sufficient.

If using ethanol, then it must be followed by flaming.

Alternatively, it should be placed in a 10% bleach solution for a few minutes, then rinsed in two sequential beakers of water. We have multiple Tissue Tearers, so the bleaching can be performed without slowing down the workflow too much - just use clean Tissue Tearer while one is bleaching.

kaitlynrm commented 6 years ago

It might be worthwhile to note that no more than 0.02 should be used as well.

yaaminiv commented 6 years ago

@kubu4 does the rinse have to be done with nanopure water, or just DI water?

kubu4 commented 6 years ago

DI's fine.

On Wed, Sep 26, 2018, 17:53 Yaamini Venkataraman notifications@github.com wrote:

@kubu4 https://github.com/kubu4 does the rinse have to be done with nanopure water, or just DI water?

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yaaminiv commented 6 years ago

Final DNA yield (will update with Bioanalyzer results later today):

https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part9/

yaaminiv commented 5 years ago

This protocol seems promising for using MethylMiner with low amounts of starting DNA: https://www.tandfonline.com/doi/pdf/10.1080/15592294.2017.1335849

Thoughts? @kubu4 I know you also mentioned a Qiagen kit that could be good for low starting volumes?

kubu4 commented 5 years ago

Yes, there should be a Qiagen bisulfite conversion kit (and library prep?) that accepts low quantities of input DNA. Don't know what it's called; you'll have to investigate.

yaaminiv commented 5 years ago

Is it their EpiTect kit?

https://www.qiagen.com/us/shop/epigenetics/epitect-fast-bisulfite-conversion-kits/#productdetails

kubu4 commented 5 years ago

What's the DNA input range?

yaaminiv commented 5 years ago

"The EpiTect Fast DNA Bisulfite Kit can be used for conversion of 1 ng–2 µg DNA in a volume of up to 20 µl or 1–500 ng in a maximum volume of 40 µl"

kubu4 commented 5 years ago

That's the one!

yaaminiv commented 5 years ago

Would it be better to use the Qiagen kit or try and adapt the low-input protocol in the paper I linked above? I couldn't find any information in the Qiagen protocol about being able to use the low input DNA methods for downstream sequencing applications.

yaaminiv commented 5 years ago

Recap from yesterday's lab meeting (correct me if I'm wrong): it's probably best to use the Qiagen kit. @kubu4 will ask Zymo how much input DNA they need for bisulfite library preparation and sequencing/how much input DNA they take in general. Based on what we hear from Zymo I can order the necessary kits/process my extracted DNA

kubu4 commented 5 years ago

Heard from Zymo tonight:

Preferred minimum: 50ng

Absolute minimum: 1ng - no QC and can't guarantee libraries and sequencing results

They do not accept bisulfite-converted DNA

yaaminiv commented 5 years ago

The consensus is that I could do MBD with my samples and get more than the absolute minimum of 1ng for each of my samples.

I could also look into enzyme-based methods. Any suggestions on where to start there?

yaaminiv commented 5 years ago

From my understanding, I would need to first use the MethylMiner kit to enrich methylated sequences, then the EpiTect kit for bisulfite conversion. The MethylMiner kit usually as a 1% yield.

The smallest DNA extraction yield I had was 282 ng, and 1% of that would be 2.82 ng. That is still more than the input volume for the EpiTect kit, and above Zymo's absolute minimum. Is there anything else I'm forgetting to consider with this workflow?

@kubu4's last comment says that Zymo does not accept bisulfite-converted DNA...does this mean we can't use Zymo for sequencing?

kubu4 commented 5 years ago

You'll also be constructing the libraries, so you'll need to look into library kits, too. Then, we can send libraries anywhere for sequencing.

On Fri, Jan 11, 2019, 11:07 Yaamini Venkataraman <notifications@github.com wrote:

From my understanding, I would need to first use the MethylMiner kit to enrich methylated sequences, then the EpiTect kit for bisulfite conversion. The MethylMiner kit usually as a 1% yield.

The smallest DNA extraction yield I had was 282 ng, and 1% of that would be 2.82 ng. That is still more than the input volume for the EpiTect kit, and above Zymo's absolute minimum. Is there anything else I'm forgetting to consider with this workflow?

Sam's last comment says that Zymo does not accept bisulfite-converted DNA...does this mean we can't use Zymo for sequencing?

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yaaminiv commented 5 years ago

@kubu4 Something like this? Looks like it will accept the low volumes I have

yaaminiv commented 5 years ago

There is also this Qiagen kit, although it is way more expensive.

kubu4 commented 5 years ago

Either one will get the job done.

On Fri, Jan 11, 2019, 11:29 Yaamini Venkataraman <notifications@github.com wrote:

There is also this Qiagen kit https://www.qiagen.com/us/shop/sequencing/qiaseq-solutions/qiaseq-methyl-library-kit/#orderinginformation, although it is way more expensive.

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sr320 commented 5 years ago

What have we used in the past?. How were your gonad libraries made? On Fri, Jan 11, 2019 at 11:33 AM kubu4 notifications@github.com wrote:

Either one will get the job done.

On Fri, Jan 11, 2019, 11:29 Yaamini Venkataraman <notifications@github.com wrote:

There is also this Qiagen kit < https://www.qiagen.com/us/shop/sequencing/qiaseq-solutions/qiaseq-methyl-library-kit/#orderinginformation , although it is way more expensive.

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.

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yaaminiv commented 5 years ago

@sr320 Gonad libraries were made with the Pico Methyl-Seq Library Prep Kit (first link). Leaning towards using that again.