H*****e
发帖数: 120 | 1 Hi experts:
I want to explore epigenome direction with some fund left in my budget.
After talking with BGI about epigenome sequencing, I prefer to start with
DNA methylation. CHIP would be too technique for us as a fresh hand. BGI
provides a low cost one called RRBS. The price is very attractive. However,
I worry for the future publication and grant application using these data.
In practice, RRBS is what we can handle because we are not able to handle
the gene desert. On paper, we try to cover all holes since reviewers are
very tough. Coverage is an issue that we can solve in the future. Anyone
familiar with it know any other weakness about it?
Many thanks! | A******u
发帖数: 61 | 2 用eRRBS啊，增强型RRBS 方法见Stadler et al., 2011
我们的实际操作中
70％ reads能map回reference genome
能覆盖90% CGI (10X seq)
40% gene promoter
20-60% LMR／UMR
我的理解，弱点自然是如果DNA 甲基化的变化没在这些区域，你就看不到咯。
However,
.
【在 H*****e 的大作中提到】
: Hi experts:
: I want to explore epigenome direction with some fund left in my budget.
: After talking with BGI about epigenome sequencing, I prefer to start with
: DNA methylation. CHIP would be too technique for us as a fresh hand. BGI
: provides a low cost one called RRBS. The price is very attractive. However,
: I worry for the future publication and grant application using these data.
: In practice, RRBS is what we can handle because we are not able to handle
: the gene desert. On paper, we try to cover all holes since reviewers are
: very tough. Coverage is an issue that we can solve in the future. Anyone
: familiar with it know any other weakness about it?
| z*t
发帖数: 863 | 3 re
提个budget的
wgbs很贵，俺们实验室是一个sample一条lane以达到足够的coverage
errbs可以pool多个sample在一条lane
【在 A******u 的大作中提到】
: 用eRRBS啊，增强型RRBS 方法见Stadler et al., 2011
: 我们的实际操作中
: 70％ reads能map回reference genome
: 能覆盖90% CGI (10X seq)
: 40% gene promoter
: 20-60% LMR／UMR
: 我的理解，弱点自然是如果DNA 甲基化的变化没在这些区域，你就看不到咯。
:
: However,
: .
| H*****e
发帖数: 120 | 4 Thanks so much!
I had spent the whole afternoon reading this paper. It is very interesting
and encouraging. However, one question puzzling me is that how confident we
can say non-BBRS covered area is not as important. My worry is that, if it
is necessary, we can combine sample to d whole-genome. But in that way, we
lose the individual variation/statistical analysis power. However, if we
can spent same money to do RRBS and not much question, we could do more
samples.
When I was student, my PI bought HPLC without buying column, which we bought
later with higher price. However, we also bought too many column and some
of them were throw to trash with opening the box.
Now I like to do it good with limited fund and also wish students can get
work done as well as good publication later. You are experienced! How
positive if I ask you to do eRRBS on CGI? How much do you feel the missing
part is important? Sorry asking so much. I am really anxious to learn this.
Many thanks!
【在 A******u 的大作中提到】
: 用eRRBS啊，增强型RRBS 方法见Stadler et al., 2011
: 我们的实际操作中
: 70％ reads能map回reference genome
: 能覆盖90% CGI (10X seq)
: 40% gene promoter
: 20-60% LMR／UMR
: 我的理解，弱点自然是如果DNA 甲基化的变化没在这些区域，你就看不到咯。
:
: However,
: .
| A******u
发帖数: 61 | 5 你要是想做eRRBS，具体实验方法看
http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjour
养了细胞，买个kit，把DNA提出来。
没信心就把DNA直接寄给BGI，让他们把建库测序全给你做了，再把信息分析做了，写好
文章给你就是。
有信心，就自己拿酶切一切，bisulfite处理一下，跑个胶，切下来，用kit做个库，给
BGI测序；测序结果你自己分析。超级简单。
interesting
we
it
we
bought
some
【在 H*****e 的大作中提到】
: Thanks so much!
: I had spent the whole afternoon reading this paper. It is very interesting
: and encouraging. However, one question puzzling me is that how confident we
: can say non-BBRS covered area is not as important. My worry is that, if it
: is necessary, we can combine sample to d whole-genome. But in that way, we
: lose the individual variation/statistical analysis power. However, if we
: can spent same money to do RRBS and not much question, we could do more
: samples.
: When I was student, my PI bought HPLC without buying column, which we bought
: later with higher price. However, we also bought too many column and some
| H*****e
发帖数: 120 | 6 Sorry, I did not explain myself clear. I guess that my mind is puzzled with
too many question and try to get answer once.
I will ask BGI to do it. Since it is too expensive, with available fund, I
can only have two imperfect choices:
1. to combine samples: for example, combine all controls and all tumors. Run
two whole genome bis-sequence.
2. down to RRBS or eRRBS, to do 3 controls and 3-5 tumors.
The first one lose the statistical power; we may have trouble to publish it
in the future. However, we have a whole picture for exploration. It is
uncertain.
The second lose non-CGI. No matter how exciting or boring the result will be
. The sample repeats are sufficient for student to publish a small paper.
My student do not see what I am seeing. He wants to do whole genome. Funding
is limited. If other project needs money, he may not have money to do it
again. So I prefer RRBS. Then, I also worry how important the non-covered
area is. If you are in this situation, which one you choose and why?
Many thanks!
【在 A******u 的大作中提到】
: 你要是想做eRRBS，具体实验方法看
: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjour
: 养了细胞，买个kit，把DNA提出来。
: 没信心就把DNA直接寄给BGI，让他们把建库测序全给你做了，再把信息分析做了，写好
: 文章给你就是。
: 有信心，就自己拿酶切一切，bisulfite处理一下，跑个胶，切下来，用kit做个库，给
: BGI测序；测序结果你自己分析。超级简单。
:
: interesting
: we
| A******u
发帖数: 61 | 7 那肯定是eRRBS了。
其实你们可以自己建库，只给BGI测序就行。
测序结果也可以自己分析的，不难。
with
I
Run
it
【在 H*****e 的大作中提到】
: Sorry, I did not explain myself clear. I guess that my mind is puzzled with
: too many question and try to get answer once.
: I will ask BGI to do it. Since it is too expensive, with available fund, I
: can only have two imperfect choices:
: 1. to combine samples: for example, combine all controls and all tumors. Run
: two whole genome bis-sequence.
: 2. down to RRBS or eRRBS, to do 3 controls and 3-5 tumors.
: The first one lose the statistical power; we may have trouble to publish it
: in the future. However, we have a whole picture for exploration. It is
: uncertain.
|
|
