Description of genome and assembly desires
SMRT Portal 2.1.1 was released within the last couple of months (packaged with Celera Assembler 7); the Celera Assembler has, in the last week, released version 8.1. This will serve as a log of my successes and failures at installing and using these two packages together to assemble a single-chromosome bacterial (circular) genome with data from a 5K PB library sequenced with a single SMRT cell; a 1/4 plate 454 from its own library prep; along with various Sanger-derived sequences. The genome in question is has long repeats (12 loci and 15 copies of one transposable element; 2 copies of another).
System: single laptop running Ubuntu 12.04 x64; 4 CPUs; dash shell
Installation
Main package versions
leading notes: I'm glad the "SEYMOURHOME" convention is being phased out in favor of SMRT_ROOT; Ubuntu doesn't have an /opt directory by default.
I followed the suggestion of adding a smrtanalysis user to my system; this had several complications
commands:
Usage review/notes/annoyances and bugs
SMRT Portal 2.1.1 was released within the last couple of months (packaged with Celera Assembler 7); the Celera Assembler has, in the last week, released version 8.1. This will serve as a log of my successes and failures at installing and using these two packages together to assemble a single-chromosome bacterial (circular) genome with data from a 5K PB library sequenced with a single SMRT cell; a 1/4 plate 454 from its own library prep; along with various Sanger-derived sequences. The genome in question is has long repeats (12 loci and 15 copies of one transposable element; 2 copies of another).
System: single laptop running Ubuntu 12.04 x64; 4 CPUs; dash shell
Installation
Main package versions
leading notes: I'm glad the "SEYMOURHOME" convention is being phased out in favor of SMRT_ROOT; Ubuntu doesn't have an /opt directory by default.
I followed the suggestion of adding a smrtanalysis user to my system; this had several complications
- my standard user account didn't have access to the dropbox directory, nor other program-related entries. At least initially, the smrtanalysis user has equal administrative permissions as my standard user account, which at least partially defeats the purpose of compartmentalization.
- the smrtanalysis user didn't have the same accesses to mysql as my main user
commands:
sudo adduser smrtanalysis #note: this adds the user to the main ubuntu login screen.
sudo apt-get install krb5-user #(unlisted depend - I believe without this you cannot create the administrator user in SMRTportal)
sudo usermod -a -G www-data smrtanalysis #(otherwise it cannot interface with a localized apache server)
sudo usermod -a -G smrtanalysis pag #(so my main user will have access to look at the directories - at least it SHOULD after I reboot)
sudo visudo #or sudo usermod -a G admin smrtanalysis #to add installation privileges to smrtanalysis user
mysql
More installation notes:sudo apt-get install krb5-user #(unlisted depend - I believe without this you cannot create the administrator user in SMRTportal)
sudo usermod -a -G www-data smrtanalysis #(otherwise it cannot interface with a localized apache server)
sudo usermod -a -G smrtanalysis pag #(so my main user will have access to look at the directories - at least it SHOULD after I reboot)
sudo visudo #or sudo usermod -a G admin smrtanalysis #to add installation privileges to smrtanalysis user
mysql
CREATE USER 'smrtanalysis'@'%'
GRANT CREATE ON *.* to 'smrtanalysis'@'%';
GRANT CREATE ON *.* to 'smrtanalysis'@'localhost';
GRANT ALL PRIVILEGES on *.* to 'smrtanalysis'@'localhost' WITH GRANT OPTION; #some scripts fail without this
GRANT ALL PRIVILEGES on *.* to 'smrtanalysis'@'%' WITH GRANT OPTION;
FLUSH PRIVILEGES;
exit
su smrtanalysisGRANT CREATE ON *.* to 'smrtanalysis'@'%';
GRANT CREATE ON *.* to 'smrtanalysis'@'localhost';
GRANT ALL PRIVILEGES on *.* to 'smrtanalysis'@'localhost' WITH GRANT OPTION; #some scripts fail without this
GRANT ALL PRIVILEGES on *.* to 'smrtanalysis'@'%' WITH GRANT OPTION;
FLUSH PRIVILEGES;
exit
SMRT_ROOT=/opt/smrtanalysis #as smrtanalysis
sudo mkdir -p $SMRT_ROOT
sudo mkdir -p /tmp/smrtanalysis #without this directory the install script will not run! unsure if sudo needed
sudo chown -R smrtanalysis:smrtanalysis $SMRT_ROOT #otherwise owned by root on my system
sudo chown -R smrtanalysis:smrtanalysis /tmp/smrtanalysis
sudo mv smrtanalysis-2.1.* /opt
cd /opt
bash smrtanalysis-2.1.1-ubuntu-12.04.run --rootdir $SMRT_ROOT # --no-extract #after the first use
(edited some setup scripts to use smrtanalysis or smrtportal user in mysql as well)
cd $SMRT_ROOT
sudo chmod -R ug+rX current/smrtanalysis-2.1.1.128549/ #so my standard user can at least get in (x) and see listings (r) of the subdirectories
sudo chmod ug+x /opt/smrtanalysis/current/etc/setup.sh
cd /opt/smrtanalysis/admin
./tomcatd start #this works under either user
./kodosd start #this would not run under non-smrtanalysis account
#installing current version of wgs...
sudo mv $DOWNLOADPATH/wgs-8.1-Linux-amd64.tar.bz2 $SMRT_HOME/install/smrtanalysis-2.1.1.128549/analysis/bin
cd $SMRT_HOME/install/smrtanalysis-2.1.1.128549/analysis/bin
sudo tar xjf wgs-8.1-Linux-amd64.tar.bz2 #creates wgs-8.1 directory and fills it
mv wgs-7.0/ wgs-7.0.0 #or wgs.old
sudo ln -s wgs wgs-7.0 #points files that depend on wgs7.0 in the path to a generic "wgs" which will be the current version. hopefully pacbio will eventually switch all files to point to a generic/current wgs directory
sudo ln -s wgs-8.1/ wgs #points the generic to the most up-to-date
sudo chown -h smrtanalysis:smrtanalysis wgs* #changes links' ownership
sudo chown -R smrtanalysis:smrtanalysis wgs-8.1/
sudo chmod -R ug=rX,o-rwx wgs-8.1/
(remove the tar.bz2 if desired)
sudo mkdir -p $SMRT_ROOT
sudo mkdir -p /tmp/smrtanalysis #without this directory the install script will not run! unsure if sudo needed
sudo chown -R smrtanalysis:smrtanalysis $SMRT_ROOT #otherwise owned by root on my system
sudo chown -R smrtanalysis:smrtanalysis /tmp/smrtanalysis
sudo mv smrtanalysis-2.1.* /opt
cd /opt
bash smrtanalysis-2.1.1-ubuntu-12.04.run --rootdir $SMRT_ROOT # --no-extract #after the first use
(edited some setup scripts to use smrtanalysis or smrtportal user in mysql as well)
cd $SMRT_ROOT
sudo chmod -R ug+rX current/smrtanalysis-2.1.1.128549/ #so my standard user can at least get in (x) and see listings (r) of the subdirectories
sudo chmod ug+x /opt/smrtanalysis/current/etc/setup.sh
cd /opt/smrtanalysis/admin
./tomcatd start #this works under either user
./kodosd start #this would not run under non-smrtanalysis account
#installing current version of wgs...
sudo mv $DOWNLOADPATH/wgs-8.1-Linux-amd64.tar.bz2 $SMRT_HOME/install/smrtanalysis-2.1.1.128549/analysis/bin
cd $SMRT_HOME/install/smrtanalysis-2.1.1.128549/analysis/bin
sudo tar xjf wgs-8.1-Linux-amd64.tar.bz2 #creates wgs-8.1 directory and fills it
mv wgs-7.0/ wgs-7.0.0 #or wgs.old
sudo ln -s wgs wgs-7.0 #points files that depend on wgs7.0 in the path to a generic "wgs" which will be the current version. hopefully pacbio will eventually switch all files to point to a generic/current wgs directory
sudo ln -s wgs-8.1/ wgs #points the generic to the most up-to-date
sudo chown -h smrtanalysis:smrtanalysis wgs* #changes links' ownership
sudo chown -R smrtanalysis:smrtanalysis wgs-8.1/
sudo chmod -R ug=rX,o-rwx wgs-8.1/
(remove the tar.bz2 if desired)
Documentation on how to auto-launch tomcatd and kodosd at system startup would be appreciated
Creating desktop shortcut to smrtview sometimes produces one with a pretty icon, sometimes a .jnlp file, depending on the method of instantiation. It is unclear what happens if this link ALREADY exists in the target location. I had to override the java version used by this application to use Oracle 7 rather than my system default. As I don't always have an internet connection enabled when launching SMRTView, it is needed to go to the Oracle Java 7 Control Panel; goto advanced and change "Check for Certificates Using" to CRLs. You will probably need an internet connection the first time you use SMRTView. I am unclear how to get the individual jnlp files for a dataset to work when created in SMRTPortal (I believe it tried launching with the wrong java version).
More to follow
Creating desktop shortcut to smrtview sometimes produces one with a pretty icon, sometimes a .jnlp file, depending on the method of instantiation. It is unclear what happens if this link ALREADY exists in the target location. I had to override the java version used by this application to use Oracle 7 rather than my system default. As I don't always have an internet connection enabled when launching SMRTView, it is needed to go to the Oracle Java 7 Control Panel; goto advanced and change "Check for Certificates Using" to CRLs. You will probably need an internet connection the first time you use SMRTView. I am unclear how to get the individual jnlp files for a dataset to work when created in SMRTPortal (I believe it tried launching with the wrong java version).
More to follow
Usage review/notes/annoyances and bugs
SMRTView tooltips disappear after a couple of seconds and information is not selectable from these tooltips, nor duplicated elsewhere in the interface. Prologue information displayed for all or most BridgeMapper reads in tooltips appears to be the same as the Epilogue information (split read view for 2-piece subreads). I would prefer subreads to be "expanded" such that a single line in the SMRTView interface is for a single BridgeMapper read (and multiple subread reads be grouped and "boxed"). Currently, the interface is compressed - which means that if the view is zoomed out, multiple reads may be split across a single line, may be overlapping, and overall give a confusing view.
BridgeMapper documentation appears to be, as yet, incomplete. It is definitely a great feature - in fact the primary reason I decided to install SMRT analysis tools (rather than just portions of SMRT pipe) and the latest SMRTView.For example, the split_reads.bridgemapper file is not explained in online docs, nor is much of the information available in SMRTView. Thanks for the link rhall - still not QUITE resolving my questions, but a lot better than the documentation that I previously located.
Bridgemapper questions:
BridgeMapper documentation appears to be, as yet, incomplete. It is definitely a great feature - in fact the primary reason I decided to install SMRT analysis tools (rather than just portions of SMRT pipe) and the latest SMRTView.
Bridgemapper questions:
- Are the Score columns the BLASR score of mapping the entire subread to the indicated location (contiguously with pro, main, epi) or just with the indicated portion?
- What does percent similarity refer to (e.g. for the whole-read mapped to one location, or just that one portion internally; essentially the same question as above)
- Is there a way to display how poorly it would map if it were left as one contiguous chunk if that's not the default above? - my genome has a lot of transposable elements, as I mentioned, so slightly better mappings elsewhere may be insignificant.
- Does the file have all reads listed? there are some that appear to have neither prologue nor epilogue, so I've filtered those out in loffice
- P.S. where can I find a BLASR score equation/description? I have many "negative" scores - is it like golf or freezers where low=good?
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