j*p
发帖数: 411 | 1 攒人品，顺便回答一下 iiiir 的问题。
我们尝试过好几种不同的SNP calling的方法，包括GATK, Samtools, Varscan,
SeqGenes, 等，并且做了SNP array 作为gold standard比较各种方法的prediction
power。
从我们的经验，BWA + GATK 最好，sensitivity 和 specificity 都在95%以上。
以下是GATK 的pipeline
假设你有一个control 样品C 和一个样本样品A的pair-end sequencing，共4个文件，C
_R1.fastq, C_R2.fastq, A_R1.fastq and A_R2.fastq如何通过BWA/GATK去找样品A中
的SNPs (相对于C)
假设assembly 用的是hg19，你的BWA index 在这里：/bwa/indexes/hg19
Check this website if you have any questions:
http://seqanswers.com/wiki/How-to/exome_analysis
#s... 阅读全帖 |
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j*p
发帖数: 411 | 2 攒人品，顺便回答一下 iiiir 的问题。
我们尝试过好几种不同的SNP calling的方法，包括GATK, Samtools, Varscan,
SeqGenes, 等，并且做了SNP array 作为gold standard比较各种方法的prediction
power。
从我们的经验，BWA + GATK 最好，sensitivity 和 specificity 都在95%以上。
以下是GATK 的pipeline
假设你有一个control 样品C 和一个样本样品A的pair-end sequencing，共4个文件，C
_R1.fastq, C_R2.fastq, A_R1.fastq and A_R2.fastq如何通过BWA/GATK去找样品A中
的SNPs (相对于C)
假设assembly 用的是hg19，你的BWA index 在这里：/bwa/indexes/hg19
Check this website if you have any questions:
http://seqanswers.com/wiki/How-to/exome_analysis
#s... 阅读全帖 |
|
发帖数: 1 | 3 THE RECONSTRUCTION OF HANKOW
China's Commercial And Industrial Capital.
From The Far Eastern Review.
Ever since the fateful fire which destroyed the Chinese city of Hankow
during the Revolution of 1911-12 it has been generally known that the
Chinese Government has been desirous of rebuilding the city upon modern
lines and making other improvements in the vicinity compatible with the
importance of such a flourishing commercial and industrial center. It was,
therefore, not surprising when it ... 阅读全帖 |
|
发帖数: 1 | 4 THE RECONSTRUCTION OF HANKOW
China's Commercial And Industrial Capital.
From The Far Eastern Review.
Ever since the fateful fire which destroyed the Chinese city of Hankow
during the Revolution of 1911-12 it has been generally known that the
Chinese Government has been desirous of rebuilding the city upon modern
lines and making other improvements in the vicinity compatible with the
importance of such a flourishing commercial and industrial center. It was,
therefore, not surprising when it ... 阅读全帖 |
|
j******n
发帖数: 21641 | 5 普拉斯的诗
Ariel
Stasis in darkness.
Then the substanceless blue
Pour of tor and distances.
God’s lioness,
How one we grow,
Pivot of heels and knees! - The furrow
Splits and passes, sister to
The brown arc
Of the neck I cannot catch,
Nigger-eye
Berries cast dark
Hooks -
Black sweet blood mouthfuls,
Shadows.
Something else
Hauls me through air -
Thighs, hair;
Flakes from my heels.
White
Godiva, I unpeel -
Dead hands, dead stringencies.
And now I
Foam to wheat, a glitter of seas.
The child’s cry
Melts ... 阅读全帖 |
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l***a
发帖数: 5114 | 6 最近翻出阿西莫夫作品全集来重看，真挺有意思的，不仅囊括了他最早期那些名不见经
传的短篇，还有自己写的“创作感想”。看到TRENDS那一篇，居然发现他在1938年（当
时他还在读大学）成功预测成两件事：1）1940年第二次世界大战，2）1970年，人类登
月。
两件事都只差1年而已！
我一直觉得那个SF黄金时期的创作高峰,后来者很难翻越……
下面附TRENDS 的原文。
TRENDS
John Harman was sitting at his desk, brooding, when I entered the office
that day.
It had become a common sight, by then, to see him staring out at the Hudson,
head in
hand, a scowl contorting his face—all too common. It seemed unfair for the
little bantam
to be eating his heart out like that day after day... 阅读全帖 |
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M****e
发帖数: 70 | 7 my point is to make sure that you have transfer of good
quality and quantity of RNA onto the blotting material.
the washing stringency for your experiment is not quite
high (i.e., 42C). i think the problem is mainly due to
bad RNA or probe. if there is a lot of radioactive
nucleotides incorporated into the probe, you should
detect very hot signal after purification. denature the
probe before use. GAPDH is a housekeeping gene that is
typically used for normalization of load. i suggest you
read a |
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l*****k
发帖数: 587 | 8 pre-absorption might help you, just use tumore sample without GFP to
pre-absorb your antibody before you use it on the real tissues.
you can also increase the stringency of your experimental condition, like
increase BSA concentration or Tween concentration, wash more rigorously.
I once made affinity-purified anti-RAD51 myself, however, I can't use it for
anything since it binds to a lot of other tissues. Wish you good luck |
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p*****n
发帖数: 981 | 9 ☆─────────────────────────────────────☆
chick (Por Los Momentos Dif Ciles) 于 (Wed Jan 3 13:21:18 2007) 提到:
I'm working on the project involved something similar to Ch-IP. Recently, I
got very weird result, in some group or controls, the Non-fixation IP has
even stronger singal than the formaldehyde fixation. This could partialy be
explained by the over-crosslinking lowered the possible detecable targets.
But, obviously, I need more stringent washing condition, at least to
diminish the non- |
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z*******a
发帖数: 175 | 10 珍惜生命，use magnetic Dyna beads.
It does not matter as long as wash more than 3 times. The stringency of your
wash buffer matters. |
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m******5
发帖数: 1383 | 11 因为没有cross linking啊，很多binding在buffer条件下就挂了。 类似的比如做一些
比较弱的相互作用的co-ip， 要摸很多buffer条件，optimize到一个合适的stringency |
|
s******u
发帖数: 81 | 12 多谢！
另外的原因可能还有digestion enzyme的sequence preference.
说到co-IP, lysis buffer一般都加detergent吗？看别人的文章基本都加，可有的
interaction加了detergent几乎检测不到。
stringency |
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h******y
发帖数: 351 | 13 我的建议是，在和老板争论是否有deletion之前，利用其他的办法证实你的PCR所发现
的结果。PCR是很容易出错的，如果你能利用Southern证实homozygous的deletion确实
存在，相信你的老板是会接受你的结论的。
Constance L. Cepko实验室最近retract了G&D上的一篇文章，很好的说明了PCR的易错
性。有兴趣的可以看看。
http://genesdev.cshlp.org.mutex.gmu.edu/content/25/12/1344.long
“We are writing to clarify the interpretation of the results from our above
-mentioned paper. In this study, we used two methods to examine the
structure of RNA from the mouse Math5 (Atoh7) locus. Our initial
characterization was an analysis of the Math... 阅读全帖 |
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l**********1
发帖数: 5204 | 14 要上 R package
pls refer:
van Hijum SA, Medema MH, Kuipers OP. (2009)
Mechanisms and evolution of control logic in prokaryotic transcriptional
regulation.
Microbiol Mol Biol Rev. 73: 481-509
Abstract
A major part of organismal complexity and versatility of prokaryotes resides
in their ability to fine-tune gene expression to adequately respond to
internal and external stimuli. Evolution has been very innovative in
creating intricate mechanisms by which different regulatory signals operate
and inter... 阅读全帖 |
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l**********1
发帖数: 5204 | 15 Plus
To LZ:
just check,
>http://bcbio.wordpress.com/tag/ngs/
cited:
>Access VCF variant information
>In addition to extending the GATK through walkers and annotations you can
also utilize the extensive API directly, taking advantage of parsers and
data structures to handle common file formats. Using Clojure’s Java
interoperability, the variantcontext module provides a high level API to
parse and extract information from VCF files. To loop through a VCF file and
print the location, reference alle... 阅读全帖 |
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m******5
发帖数: 1383 | 16 目的蛋白是个transcription factor，表达很低。
看了不少Chip protocol，似乎stringency都很高，貌似都是给 histon之类表达量高的
蛋白用的，有同修接触过类似情况么? |
|
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b*****h
发帖数: 3386 | 18 【 以下文字转载自 Stock 讨论区 】
发信人: lyrist (我是捂大的), 信区: Stock
标 题: 如何做Swing trade (3) - Ascending Triangle
发信站: BBS 未名空间站 (Sun May 31 19:58:25 2009)
我们青蛙们最关心的事情是如何得到一个好pick，与其等着大牛给pick，不如自己动手
来找。
我个人在买卖股票，设置stop的时候基本只看trend, support/resistence and volume
这些东西，但在scan股票的时
候，这些咚咚不好直接定义，所以必须借助一些technical indicators。 所以， 在筛
选股票之前，我们必须了解一些基
本的technical indicators。对于筛选股票常用的technical indicators大概分为下面
几类：
1. Money flow:
大家在看蜡烛图的量的时候，会很直观的感觉，如果一段时间绿柱柱比红柱柱多，而且
高，这说明有更多的demand, 当
demand大于supply, 股价就会上涨（跟街上买白菜是一个道理... 阅读全帖 |
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