关于bisulphite genome sequencing的问题

钻石星辰

回复 sannia 的帖子7 @5 d, T+ q, T

6 p9 ^6 k) _: V( r% x) K+ S: r在生物学链接里可以看到这篇文章的部分内容（百度里查也是），可是已打开文章就是乱码了……

sannia

一．模板制备* w& J1 P2 [1 S% d
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5 Z/ w1 I: F7 ?/ Q# _3 cEZ DNA Methylation-Gold Kit (ZYMO RESEARCH, product number: D5005) （该公司曾经破产，有段时间买不到这个产品，现在貌似复活了但产品编号不知道是不是还是这个。不过busulfite sequencing 试剂盒基本都差不多）
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原理是Conversion Reagent将DNA序列中没有被甲基化的C变成T，甲基化的C不变。这样通过测序，将原序列和处理后的序列比较，就可以分析出各个位点的甲基化状况。) w$ U9 K5 W0 M& t% S+ I
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, K, j( k* R9 E0 _7 r) Z- l- ~! WThe amount of DNA per treatment can range from 500 pg to 2 μg. An optimal amount is 200-500 ng. （我用750 ng）8 j0 ^. D6 g8 s" C% m$ f2 U' K/ P
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1. Add 130 μl of the CT Conversion Reagent solution to 20 μl of your DNA sample in a PCR tube. If the volume of the DNA sample is less than 20 μl, make up the difference with water. Mix the sample by flicking the tube or pipetting the sample up and down.
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For Example: 4 μl DNA Sample, 16 μl water, 130 μl CT Conversion Reagent. b1 P( F7 i' |3 h# p

( j0 ~7 b8 D- {) e  L) r+ a$ ~2. Place the sample tube in a thermal cycler and perform the following steps*:
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1. 98°C for 10 minutes
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2. 64°C for 2.5 hours
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( q3 b8 e6 O. I* X. \3. 4°C storage up to 20 hours.  （意思好像是至多只能放20个小时，英文不好，我理解成至少了，结果问题也不大）
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*For some samples, alternative parameters may yield improved results (see Appendix). If you have been using this kit with good results using different reaction conditions than described above, you can continue using those same conditions.
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. v  e. M; ^# h: }3. Add 600 μl of M-Binding Buffer into a Zymo-Spin IC™ Column and place the column into a provided Collection Tube.
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4. Load sample (from Step 2) into the Zymo-Spin IC™ Column containing the MBinding Buffer. Close the cap and mix by inverting the column several times.) W, J( [! T" V0 `

1 n( ~2 L9 S' z# e1 Y, g5. Centrifuge at full speed (>10,000 x g) for 30 seconds. Discard the flow-through.
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8 B/ [" C3 e9 D+ y7 k$ |6. Add 200 μl of M-Wash Buffer to the column. Spin at full speed for 30 seconds.) b( E/ x) I. Q- r5 ~9 B
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7. Add 200 μl of M-Desulphonation Buffer to the column and let stand at room temperature (20 °C – 30 °C) for 15-20 minutes. After the incubation, spin at full speed for 30 seconds.9 f0 [  d/ t! I' @" x
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8. Add 200 μl of M-Wash Buffer to the column. Spin at full speed for 30 seconds.
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Add another 200 μl of M-Wash Buffer and spin at top speed for 30 seconds.; b& _1 j' N) M5 s7 v% Q

, {; ]  {7 B, V2 |/ O9. Add 10 μl of M-Elution Buffer directly to the column matrix. Place the column into a 1.5 ml tube. Spin briefly at full speed to elute the DNA. （我用50 μl 洗两次，一共得到100 μl 模板，每次取5 μl 做PCR反应）
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The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. We recommend using 2 – 4 μl of the eluted DNA for each PCR.
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! o5 A+ J; l' d* J% r- F$ G0 T$ {Reagent Preparation( Y. Q% R+ [7 ~+ y
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Before starting, prepare the CT Conversion Reagent and M-Wash Buffer as described below.
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1. Preparation of CT Conversion Reagent
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The CT Conversion Reagent supplied within this kit is a solid mixture and must be prepared prior to first use. Prepare by adding 900 μl water, 50 μl of M-Dissolving Buffer, and 300 μl of M-Dilution Buffer to a tube of CT Conversion Reagent. Dissolve at room temperature with intermittent, brief vortexing for a total of 10 minutes
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or shake the tube on a shaker for 10 minutes. It is normal to see trace amounts of undissolved reagent, since the concentration of the CT Conversion Reagent in solution is saturated. Store the CT Conversion Reagent solution away from light at room temperature (20°C – 30°C) until use. Each tube of CT Conversion Reagent is designed for ten separate DNA treatments. For best results, the CT Conversion Reagent should be used immediately following preparation. If not used immediately, the CT Conversion Reagent solution can be stored for up to one week at –20°C. Stored CT Conversion Reagent solution must be thawed at room temperature and mixed for 2 minutes by rotating or vortexing prior to use. The CT Conversion Reagent is light sensitive, so minimize its exposure to light. （使用之前室温震荡30分钟，可以用橡皮筋将管绑到振荡器上面）
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2. Preparation of M-Wash Buffer& V$ S/ V- e# T: ?7 \6 M

6 o( b7 n6 X( j: F# j2 M* {4 CAdd 24 ml (96 ml for EZ DNA Methylation-Gold Kit™ D5006 for 200 sample size) of 100% ethanol to the M-Wash Buffer Concentrate to make the final M-Wash Buffer solution.
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二．引物设计
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& s( E& r/ r5 w$ `8 Z8 thttp://katahdin.mssm.edu/kismeth/primer_design.pl
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$ {/ r+ }  A: E8 s# C序列要用fasta格式。
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# f5 e, m  R+ X$ t1 \我用的参数一般是，引物长度从23-30，PCR产物长度从200-400bp，Tm最好有50。' Z' m1 P8 p5 o' w- c

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三．PCR
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4 }# v4 S5 x! p4 i# n一般处理后的DNA GC含量较低，比较难以扩增。我使用的是sigma的P0982 JumpStart™ REDTaq® ReadyMix™ Reaction Mix。扩增条件：94, 5min; 94, 40s; 50, 40s; 72, 2min; 45 cycles. 用TAKARA LA Taq似乎也可以。
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四．比较结果+ u, `! Z% |0 B: Z7 C* B

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& d: i" v9 B( }+ k1 ^# C7 q接下来就是通常的连T-vector，测序。测序回来后的序列，去掉引物，以fasta格式排列好，存到一个文档中。把原序列去掉引物，同样以fasta格式保存为一个文档。0 ]1 b$ I8 V# Z! x# R* v! T+ z
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http://katahdin.mssm.edu/kismeth/revpage.pl6 `4 f8 s3 j+ f3 }

, a( `$ m1 `; {( @, h" i) E5 w左边输入原序列，右边输入测序回来的序列。点击分析按钮就可以得到结果，分为CG，CNG，CNN三项。（这个网站对序列格式要求比较严格，序列中不能有点，空格等）

sannia

我转过来了 你看看有没有帮助吧

钻石星辰

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回复 sannia 的帖子( y+ a/ S5 U" H2 P) m% p
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谢谢你。还有两个小问题。
6 F' Z9 g9 P) O* I1 s1. 重复10次实验是指挑了10个阳性克隆去测序吗？* X  ]1 F% O0 z* n6 s
2. 我看有些文献建议在制备模版之前最好用限制性内切酶切一下，这样有必要吗？

sannia

1. 对。但是现在很多杂志都要求15个克隆左右。7 B% H4 h% I" [/ t5 g
2. 我们没有做酶切。

钻石星辰

回复 sannia 的帖子
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是一个板子上的15个克隆？- j  c; q0 h5 K+ M  ~
如果是，那么一个PCR产物band为什么会有个别几个CpG位点差异？
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  u+ a( i" C3 M) \+ K还是从甲基化开始就做10个平行？

sannia

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是一个板上的10-20个克隆，因为你的模板是个混合物，bisulfite 盐的转化效率不是百分之百的，不是所有的非甲基化的C都能变成T。所以要你多测几个克隆，有一定统计学的意义。

hcluo

该扩展PCR片段中共有6个CG序列（横向的6个圈表示）；另外纵向的10行，表示作者作了10个重复，10个不同的样本的结果。

hcluo

黑点表示的是甲基化位点，白圈的地方表示CG没有甲基化。

钻石星辰

回复 sannia 的帖子
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! d  V( ^; I4 c% P/ k3 s9 b! F你好！我使用了您推荐的ZYMO试剂盒，效果还不错。纯化出的模板进行PCR。我使用500ng的gDNA，最后回收得到150ng左右。
* W5 f( F6 c4 M我使用的引物是Yamanaka (2006)使用的Oct4引物和Nanog引物。可是，只有Oct4扩增出了目的条带且很明亮，Nanog引物没有扩出来。请问是模板的问题还是引物的问题呢？接下来我应该先优化退火温度还是甲基化条件呢？
& v! e+ `5 L* ?0 k  j% g- g谢谢