十年的生信

免疫荧光实验

发表于2019年1月5日由daizao

撒云俐实验笔记
* 1：6孔板种细胞并转染 (6 x 10^5 cell/孔）
* 2：36h后，将细胞种到12孔板中，(种细胞前，用灭过菌的镊子将细胞怕片放到12孔板中，种2 x 10^5 cell/孔)
* 3：待细胞完全贴壁值 70%~80% 后，加药处理24h
* 4：24h后，沿培养基12孔板壁加 1 x $ PBS(预先恢复至室温)，清洗3遍
* 5：弃1 x PBS，加 4% 多聚甲醛，室温静置固定20分钟，1 x PBS 洗2次（5分钟/次）
* 6：加0.5% TritonX-100，1ml/孔，室温静置20分钟。1 x PBS洗3次，3分钟/次
* 7：封闭：去除玻片，正面朝上放到另一新的12孔板中，每孔加800微升3%BSA封闭1h
* 8：孵一抗：吸去封闭液，每片滴加足量稀释的一抗（用0.1%BSA配置），加盖湿盒，4摄氏度过夜孵育（一抗稀释比详见说明书）
* 9：吸去一抗，每孔加1mL 1 x PBS清洗，放在水平摇床上清洗，5分钟/次（共3次），孵二抗（二抗用之前离心，不要将沉淀吸出），室温避光孵育1小时（1：400）荧光二抗，用0.1%BSA配置，PBST清洗3次（3分钟/次）
* 10（复染核）：滴加DAPI（1mg/ml）避光孵育5分钟后，PBST洗4次，5min/次
* 11：（封片拍照）：将玻片正面向下倒扣在载玻片上，四周用指甲油固定，待干涸后，可现在普通荧光显微镜下观察是否有荧光，再用激光共聚焦显微镜观察由于wordpress的markdown不好用，附一个用印象笔记写的链接：

撒云俐实验笔记

发表在实验操作 | 留下评论

TCGA和dbGaP申请中的注意事项（SAM更新是否需要缴费）

发表于2019年1月5日由daizao

SAM的renew不用交费，所以收到的所有关于缴费的邮件都是假的！

发表在 TCGA | 留下评论

GATK4_Spark版本_虚拟机并行计算测试

发表于2018年12月19日由daizao

GATK4架构总览

Spark_Hadoop的GATK4，在后台自动进行了数据的切割

发表在 Linux | 留下评论

GATK_FPGA加速器驱动安装

发表于2018年9月27日由daizao

#Installing Required OS Packages and Components
sudo yum groupinstall 'Development and Creative Workstation'
sudo yum groupinstall 'Additional Develpment'
sudo yum groups mark install "Compatibility Libraries"
sudo yum groupinstall 'Development Tools' --setopt=group_package_types=mandatory,default,optional
sudo yum groupinstall 'Platform Development'
sudo yum groupinstall 'Python'
sudo yum groups mark install "Virtualization Hypervisor"

#########
sudo dmidecode -t bios  
cat /proc/cpuinfo   
cat /etc/redhat-release 
cat /proc/version   
#########

tar -zxf a10_gx_pac_ias_1_1_prq_rte_installer.tar.gz -C /opt/sysoft/
cd /opt/sysoft/a10_gx_pac_ias_1_1_prq_rte_installer/
mkdir /opt/sysoft/intelrtestack_1.1
./setup.sh

cd ../intelrtestack_1.1/
source init_env.sh

wget -c https://www.intel.com/content/dam/altera-www/global/en_US/others/solutions/acceleration-hub/a10_gx_pac_ias_1_1_pv_eth.patch
cp a10_gx_pac_ias_1_1_pv_eth.patch a10_gx_pac_ias_1_1_pv/hw/
cd a10_gx_pac_ias_1_1_pv/hw/
patch -s -p0 < a10_gx_pac_ias_1_1_pv_eth.patch 

#安装OPAE Framework 
sudo yum install gcc gcc-c++ cmake make autoconf automake libxml2 libxml2-devel json-c-devel boost ncurses ncurses-devel ncurses-libs boost-devel libuuid libuuid-devel python2-jsonschema doxygen hwloc-devel libpng12 rsync
#1 Install the FPGA driver
  a Remove any previous version of the OPAE framework
    sudo yum remove opae*.x86_64
  b Install the Extra Packages for Enterprise Linux (EPEL):
    #sudo yum install epel-release
  c Change to the OPAE installation software directory:
    cd /opt/sysoft/intelrtestack_1.1/a10_gx_pac_ias_1_1_pv/sw/
  d Install the driver:
    sudo yum install opae-intel-fpga*.rpm
#2 Install the latest OPAE framework:
    sudo yum install opae*.rpm
#3 Update dynamic linker run-time bindings:
    sudo ldconfig
#4 Check the Linux kernel installation:
    lsmod | grep fpga

#5 Verify your library installation:
    ls /usr/lib64/libopae-c* -l
#Run the fpgainfo tool to identify the FIM currently loaded
    sudo fpgainfo fme
cd ..
rm -rf a10_gx_pac_ias_1_1_pv_rte_installer/

发表在 Linux | 留下评论

WashU_install

发表于2018年9月20日由daizao

pip install SOAPpy
sudo service mysqld start
sudo yum install libpng ghostscript GMT hdf5
sudo yum install zlib-devel ncurses-devel
sudo yum install openssl-devel
sudo yum install openssl-devel
sudo yum install curl-devel
sudo yum install libssh2-devel
sudo yum install librsvg2-devel
tar -xf /home/train/software/libpng-1.2.59.tar.xz -C /home/train/
cd
cd libpng-1.2.59/
mkdir /opt/sysoft/libpng12
./configure --prefix=/opt/sysoft/libpng12/
make -j 8
make install
echo 'export LD_LIBRARY_PATH=/opt/sysoft/libpng12/lib/:$LD_LIBRARY_PATH' >> ~/.bashrc.WashU
echo 'export C_INCLUDE_PATH=/opt/sysoft/libpng12/include/:$C_INCLUDE_PATH' >> ~/.bashrc.WashU
source ~/.bashrc.WashU
sudo vim /etc/httpd/conf/httpd.conf
<IfModule mpm_worker_module>
   LoadModule cgid_module modules/mod_cgid.so
</IfModule>
<IfModule mpm_event_module>
   LoadModule cgid_module modules/mod_cgid.so
</IfModule>
<IfModule mpm_prefork_module>
   LoadModule cgi_module modules/mod_cgi.so
</IfModule>
sudo service httpd start
cd
rm -rf libpng-1.2.59/
mkdir WashU
cd WashU/
git clone https://github.com/epgg/eg.git

#Compile the cgi-bin program
cd eg/cgi-bin/jsmn-example/
sudo vim /etc/httpd/conf/httpd.conf
LoadModule cgid_module modules/mod_cgid.so
Options FollowSymLinks ExecCGI
AddHandler cgi-script .cgi

sudo yum install jsoncpp-devel
scp root@192.168.199.220:/root/Downloads/curl-7.61.1.tar.gz /home/train/software/
tar -zxf /home/train/software/curl-7.61.1.tar.gz -C /home/train/
cd /home/train/curl-7.61.1/
mkdir /opt/sysoft/curl-7.61.1
./configure --prefix=/opt/sysoft/curl-7.61.1/
make -j 8
make test
make install
echo 'export LD_LIBRARY_PATH=/opt/sysoft/curl-7.61.1/lib/:$LD_LIBRARY_PATH' >> ~/.bashrc.WashU
echo 'export C_INCLUDE_PATH=/opt/sysoft/curl-7.61.1/include/:$C_INCLUDE_PATH' >> ~/.bashrc.WashU
echo 'export PATH=/opt/sysoft/curl-7.61.1/bin/:$PATH' >> ~/.bashrc.WashU
source ~/.bashrc.WashU
cd ..
rm -rf curl-7.61.1/
cd /home/train/WashU/eg/cgi-bin/jsmn-example
make
cd ../jsmn/
make 
cd ..
cp subtleKnife.c subtleKnife.c.bak
vim subtleKnife.c
#define WWWT "/var/www/html/browser/t" // Docker version
#define BINdir "."
#define Mysqlserver "localhost" // Deployment version
#define Mysqlserver "localhost" // Docker version
#define Mysqluser "hguser"
#define Mysqlpswd "hguser"
cp postdeposit.c postdeposit.c.bak
vim postdeposit.c
#define WWWT "/var/www/html/browser/t" // Docker version
make 
sudo cp postdeposit subtleKnife /var/www/cgi-bin/
sudo cp ucsc2jsonhub.py /var/www/cgi-bin/

#Directories
sudo mkdir -p /var/www/html/browser/t
sudo mkdir /srv/epgg
sudo chown apache.apache /srv/epgg/
sudo mkdir /srv/epgg/data
cd /srv/epgg/data/
sudo mkdir data
cd data/
sudo mkdir subtleKnife
cd subtleKnife/
sudo mkdir seq
sudo mkdir /srv/epgg/data/data/subtleKnife/hg19
sudo mkdir /srv/epgg/data/data/subtleKnife/hg19/config
sudo mkdir /srv/epgg/data/data/subtleKnife/hg19/session
sudo mkdir /srv/epgg/data/trash
sudo chown apache.apache /srv/epgg/data/trash/
sudo ln -s /srv/epgg/data/trash /usr/lib

cd ~/WashU/eg/
cd browser/
sudo mv css/ index.html js/ images/ /var/www/html/browser/
cd ..
sudo mv config/treeoflife /srv/epgg/data/data/subtleKnife/
sudo cp config/hg19/tracks.json /srv/epgg/data/data/subtleKnife/hg19/config/
sudo cp config/hg19/publichub.json /srv/epgg/data/data/subtleKnife/hg19/config/
cd /srv/epgg/data/data/subtleKnife/hg19/
sudo vim hg19_download.sh
####################################
wget -c http://egg.wustl.edu/d/hg19/refGene.gz
wget -c http://egg.wustl.edu/d/hg19/refGene.gz.tbi
wget -c http://egg.wustl.edu/d/hg19/gencodeV17.gz
wget -c http://egg.wustl.edu/d/hg19/gencodeV17.gz.tbi
wget -c http://egg.wustl.edu/d/hg19/xenoRefGene.gz
wget -c http://egg.wustl.edu/d/hg19/xenoRefGene.gz.tbi
####################################
sudo bash hg19_download.sh
sudo rm hg19_download.sh

mysql -u root -p123456
mysql> GRANT ALL ON *.* TO 'hguser'@'localhost' IDENTIFIED BY 'hguser' WITH GRANT OPTION;
mysql> FLUSH PRIVILEGES;
mysql> CREATE DATABASE hg19;
mysql> FLUSH PRIVILEGES;
mysql> QUIT
cd ~/WashU/eg/config/
cat sessionUtils.sql | mysql -u hguser -p hg19
cd hg19
cat makeDb.sql | mysql -u hguser -p hg19
sudo vim /etc/httpd/conf/httpd.conf
#############################
<Location /var/www/cgi-bin>
Header set Access-Control-Allow-Origin "*"
</Location>
#############################
sudo service httpd restart
sudo service mysqld restart

cd /srv/epgg/data/data/subtleKnife/seq/
sudo wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19.gz http://epgg-test.wustl.edu/installdata/hg19.gz.tbi

cd
mkdir -p ~/WashU/webdata/hg19
cd ~/WashU/webdata/hg19/
vim download.sh
##########################################################
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/rmsk_ensemble.gz
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/rmsk_ensemble.gz.tbi
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/rmsk_all.gz
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/rmsk_all.gz.tbi
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/cpgisland.gz
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/cpgisland.gz.tbi

wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/rmsk.tgz

wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/gc5Base.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/placentalphastCons46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/placentalphastCons46way.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg19/placentalphyloP46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/placentalphyloP46way.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg19/primatephastCons46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/primatephastCons46way.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg19/primatephyloP46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/primatephyloP46way.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg19/vertebratephastCons46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/vertebratephastCons46way.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg19/vertebratephyloP46way.gz \
http://epgg-test.wustl.edu/installdata/hg19/vertebratephyloP46way.gz.tbi

wget -c -b -q http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign100mer.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign24mer.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign36mer.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign40mer.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign50mer.bigWig \
http://epgg-test.wustl.edu/installdata/hg19/wgEncodeCrgMapabilityAlign75mer.bigWig
##########################################################################################
tar zxvf rmsk.tgz
sudo ln -sv /home/train/WashU/webdata/hg19/* /srv/epgg/data/data/subtleKnife/hg19/
cd /srv/epgg/data/data/subtleKnife/
sudo wget http://epgg-test.wustl.edu/installdata/hg19/sql.tgz
sudo tar -zxf sql.tgz 
sudo rm sql.tgz
cd hg19/
cat load.sql | mysql -u hguser -p hg19
cd ~/WashU/eg/config/hg19/
cat makeDb.sql ../sessionUtils.sql | mysql -u hguser -p hg19

cd /srv/epgg/data/data/subtleKnife/
sudo mkdir hg38
cd hg38/
sudo vim download.sh
#######################################
wget -c -b -q http://epgg-test.wustl.edu/installdata/hg38/gc5Base.bigWig \
http://epgg-test.wustl.edu/installdata/hg38/refGene.gz \
http://epgg-test.wustl.edu/installdata/hg38/refGene.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg38/xenoRefGene.gz \
http://epgg-test.wustl.edu/installdata/hg38/xenoRefGene.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg38/rmsk_all.gz \
http://epgg-test.wustl.edu/installdata/hg38/rmsk_all.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg38/rmsk_ensemble.gz \
http://epgg-test.wustl.edu/installdata/hg38/rmsk_ensemble.gz.tbi \
http://epgg-test.wustl.edu/installdata/hg38/rmsk.gz \
#######################################
cd
cd WashU/webdata/
mkdir hg38
cd hg38
sudo mv /srv/epgg/data/data/subtleKnife/hg38/download.sh /home/train/WashU/webdata/hg38/
bash download.sh
wget -c https://epgg-test.wustl.edu/installdata/hg38/rmsk_ensemble.gz.tbi
wget -c https://epgg-test.wustl.edu/installdata/hg38/rmsk.tgz
tar -zxf rmsk.tgz
cd /srv/epgg/data/data/subtleKnife/hg38/
sudo mkdir config
sudo mkdir session
sudo chown apache.apache /srv/epgg/data/data/subtleKnife/hg38/session/
sudo cp ../hg19/config/publichub.json config/
sudo ln -sv /home/train/WashU/webdata/hg38/* /srv/epgg/data/data/subtleKnife/hg38/
sudo wget http://epgg-test.wustl.edu/installdata/hg38/sql.gz
sudo gunzip sql.gz
sudo mysql -u root -p123456
mysql> CREATE DATABASE hg38;
mysql> FLUSH PRIVILEGES;
mysql> quit
cat sql | mysql -u hguser -p hg38
cd ../seq/
sudo wget -c -b -q http://epgg-test.wustl.edu/installdata/hg38.gz http://epgg-test.wustl.edu/installdata/hg38.gz.tbi

cd
cd software/
wget http://hgdownload.soe.ucsc.edu/admin/jksrc.zip
cp jksrc.zip /home/train/
unzip jksrc.zip -d /opt/sysoft/
cd /opt/sysoft/kent/src/
export MACHTYPE="x86_64"
export USE_SSL=1
sudo yum install uuid-devel.x86_64
sudo yum install uuid-c++-devel.x86_64
sudo yum install libuuid-devel
make libs
mkdir utils/bwquery
cd utils/bwquery/
cp ~/WashU/eg/cgi-bin/bwquery/* .
make 
sudo ln -sv /opt/sysoft/kent/src/utils/bwquery/ /var/www/cgi-bin/
/var/www/cgi-bin/bwquery/bwquery https://vizhub.wustl.edu/hubSample/hg19/sample.bigWig chr7 27053199 27373567 20 /opt/sysoft/kent/src/utils/bwquery/bwOutput.txt 1
sudo cp bwOutput.txt /srv/epgg/data/data/subtleKnife/hg19/

cd /srv/epgg/data
sudo tar -xf /home/train/software/samtools-0.1.19.tar.bz2 -C /srv/epgg/data/
cd samtools-0.1.19/
sudo make 
sudo ln -sv /srv/epgg/data/samtools-0.1.19/samtools /usr/local/bin/
cd /opt/sysoft/
mkdir -p subtleKnife/scripts/bamliquidator
cd subtleKnife/scripts/bamliquidator/
wget -c https://egg.wustl.edu/script/bamliquidator/bamliquidator.c
wget https://egg.wustl.edu/script/bamliquidator/bamliquidator.o
wget https://egg.wustl.edu/script/bamliquidator/make.sh
chmod 755 make.sh 
sudo ./make.sh 
cp bamliquidator /usr/local/bin/
sudo yum install sendmail-devel

cd /srv/epgg/data/
sudo tar -xf /home/train/software/tabix-0.2.6.tar.bz2 -C /srv/epgg/data/
cd tabix-0.2.6/
sudo make 
sudo ln -sv /srv/epgg/data/tabix-0.2.6/tabix /usr/local/bin/

发表在 3D_genome | 留下评论

Rosetta_install

发表于2018年9月8日由daizao

source ~/.bashrc.gcc-4.9.3
mkdir /opt/sysoft/llvm_clang-3.4.2
cd /home/train/data/software/Clang
tar -zxf cfe-3.4.2.src.tar.gz
tar -zxf clang-tools-extra-3.4.src.tar.gz
tar -zxf compiler-rt-3.4.src.tar.gz
tar -zxf dragonegg-3.4.src.tar.gz
tar -zxf libcxx-3.4.2.src.tar.gz
tar -zxf lld-3.4.src.tar.gz
tar -zxf lldb-3.4.src.tar.gz
tar -zxf llvm-3.4.2.src.tar.gz
tar -zxf polly-3.4.src.tar.gz
tar -zxf test-suite-3.4.src.tar.gz
mkdir LLVM_3.4.2
mv *.gz LLVM_3.4.2
mv llvm-3.4.2.src/ llvm
mv cfe-3.4.2.src/ clang
mv clang/ llvm/tools/
mv clang-tools-extra-3.4/ extra
mv extra/ llvm/tools/clang/tools/
mv compiler-rt-3.4/ compiler-rt
mv compiler-rt/ llvm/projects/
mv lld-3.4/ lld
mv lld llvm/tools/
mv polly-3.4/ polly
#mv polly/ llvm/tools/
mv libcxx-3.4.2.src/ libcxx
mv libcxx/ llvm/projects/
mv test-suite-3.4/ test-suite
mv test-suite/ llvm/projects/
mv lldb-3.4/ lldb
#mv lldb/ llvm/tools/
mv dragonegg-3.4/ dragonegg
mkdir build
cd build/
../llvm/configure --prefix=/opt/sysoft/llvm_clang-3.4.2/ --enable-optimized --enable-assertions --enable-targets=host-only
#若出现isl required but not found,则说明需要安装isl
wget -c http://isl.gforge.inria.fr/isl-0.13.tar.gz
tar -zxf isl-0.13.tar.gz
cd isl-0.13
mkdir /opt/sysoft/isl-0.13
./configure --prefix=/opt/sysoft/isl-0.13/
make -j 8 && make install
sudo ln -sv /opt/sysoft/isl-0.13/include/isl /usr/include/isl-0.13
../llvm/configure --prefix=/opt/sysoft/llvm_clang-3.4.2/ --enable-optimized --enable-assertions --enable-targets=host-only --with-isl=/opt/sysoft/isl-0.13/ --with-cuda=/usr/local/cuda-9.2/
#若出现unrecognized options: --enable-optimized, --enable-assertions, --enable-targets,因为不可以将polly文件夹先放入llvm的tools文件夹下
#../llvm/configure --prefix=/opt/sysoft/llvm_clang-3.4.2/ --with-isl=/opt/sysoft/isl-0.13/ --with-cuda=/usr/local/cuda-9.2/
make -j 10
make check-all
make install
sudo vim /etc/ld.so.conf.d/llvm_clang-3-4-2.conf
#写入如下
/opt/sysoft/llvm_clang-3.4.2/lib/
sudo ldconfig
sudo ln -sv /opt/sysoft/llvm_clang-3.4.2/include/* /usr/include/
sudo ln -sv /opt/sysoft/llvm_clang-3.4.2/include/c++ /usr/include/llvm-clang-c++
echo 'PATH=$PATH:/opt/sysoft/llvm_clang-3.4.2/bin/' >> ~/.bashrc
source ~/.bashrc
source ~/.bashrc.gcc-4.9.3
clang --version
rm -rf build/ llvm/

tar -zxf scons-3.0.1.tar.gz
mkdir /opt/sysoft/SCons-3.0.1
python setup.py install --prefix=/opt/sysoft/SCons-3.0.1/
ln -sv /opt/sysoft/SCons-3.0.1/lib/scons-3.0.1/SCons /opt/sysoft/Python-2.7.11/lib/python2.7/site-packages/
echo 'PATH=$PATH:/opt/sysoft/SCons-3.0.1/bin/' >> ~/.bashrc
source ~/.bashrc
cd ..
rm -rf scons-3.0.1
mkdir /opt/biosoft/rosetta-3.9
tar -zxf rosetta_src_3.9_bundle.tgz -C /opt/biosoft/rosetta-3.9
cd /opt/biosoft/rosetta-3.9/rosetta_src_2018.09.60072_bundle/
cd main/source/
export PATH=/usr/lib64/openmpi/bin/:$PATH
./scons.py -j 20 bin mode=release extras=mpi
echo 'PATH=$PATH:/opt/biosoft/rosetta-3.9/rosetta_src_2018.09.60072_bundle/main/source/bin/' >> ~/.bashrc
source ~/.bashrc

mkdir /opt/biosoft/PyRosetta4-python27
tar -xf PyRosetta4.Release.python27.linux.release-189.tar.bz2 -C /opt/biosoft/PyRosetta4-python27/
cd /opt/biosoft/PyRosetta4-python27/PyRosetta4.Release.python27.linux.release-189/
cd setup/
source ~/.bashrc_local_python_2.7
python setup.py install
#不可以设置路径，最终安装到的路径为
#/opt/sysoft/Python-2.7.11/lib/python2.7/site-packages/pyrosetta-2018.34+release.14e0a7a-py2.7-linux-x86_64.egg/

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