[rskr]

The recprical BLAST might have merit, have you tried on two gold standard species like mouse and human, I'd like to see what gives for a quality score on those.

[Blahah404]

The recprical BLAST might have merit, have you tried on two gold standard species like mouse and human, I'd like to see what gives for a quality score on those.

Are you asking me? Yes we have extensively validated it - although we have a somewhat more nuanced approach than just a reciprocal best blast. I'll explain more when the paper is out (currently under review).

I work with plant data mostly. To give you an idea of the sensitivity, the number of RBB hits can range from fewer than 1000 hits to the Rice proteome (single representitive protein for each locus) for a bad assembly of a somewhat related genus like Echinochloa or Cyperus, and for those same genera a really good assembly will get up to about 15-17000 RBB hits. It's a sensitive metric, and it selects against fragmentation.

I'll run mouse v human now.

[kp5091]

Hey Blahah404,

Could you help me how to run reciprocal best-hit blast with my transcriptome?
I tried to find how to run RBH since our discussion, however, I failed to find details how to run it.

[Blahah404]

hi kp5091

I put together a quick program called transrate today to analyse transcriptome assemblies. At the moment it just gives you various metrics on a single assembly. There will be a more full version later which allows comparison of multiple assemblies and an overall quality score.

It's on Github here - I hope the instructions are clear. I'll update with more explanation of the metrics soon. Please read the installation instructions, and if you have questions it would be best to open a new thread in the de-novo assembly forum.

[kp5091]

Hi Blahah404,

At first, thank you for your help to provide good program 'transrate'.
I tried to install transrate on my macbook pro, however, I was not able to install it because version of Ruby was lower than 1.9.2. So I downloaded RVM and other programs and install higher version of Ruby after overcoming several barriers. (As I told, all my fingers is thumb in computer. ). Also, I finished installation of transcrate on my macbook pro.
Now, it's time to install Usearch and Bowtie2. I downloaded Usearch for Unix and MacOS. However, I do not have idea how to install it on my macbook pro. Unfortunately, Usearch installation site says that "if you do not know how to install it, you would better ask to colleague who is familiar to computer language. So, could you please help me to how to install Usearch and Bowtie2 on my macbook pro?
Many thanks in advance.

Andy

[luc]

I guess, you might be better off finding a linux box.

Hi Blahah404,

At first, thank you for your help to provide good program 'transrate'.
I tried to install transrate on my macbook pro, however, I was not able to install it because version of Ruby was lower than 1.9.2. So I downloaded RVM and other programs and install higher version of Ruby after overcoming several barriers. (As I told, all my fingers is thumb in computer. ). Also, I finished installation of transcrate on my macbook pro.
Now, it's time to install Usearch and Bowtie2. I downloaded Usearch for Unix and MacOS. However, I do not have idea how to install it on my macbook pro. Unfortunately, Usearch installation site says that "if you do not know how to install it, you would better ask to colleague who is familiar to computer language. So, could you please help me to how to install Usearch and Bowtie2 on my macbook pro?
Many thanks in advance.

Andy

[Blahah404]

Andy,

I've written up an installation tutorial for linux/unix beginners here: http://blahah.net/bioinformatics/201...ing-transrate/

Please post a comment at the bottom of the tutorial if you have any trouble.

Richard

[luc]

Thanks a lot Richard!
That looks very helpful and would deserve it's own thread.

Andy,

I've written up an installation tutorial for linux/unix beginners here: http://blahah.net/bioinformatics/201...ing-transrate/

Please post a comment at the bottom of the tutorial if you have any trouble.

Ricahrd

[Blahah404]

Thanks luc - I'll make a new thread announcing transrate once I've tidied up the documentation and fixed a few minor bugs.

Andy, I actually agree with luc that you should try using linux. You *can* run most bioinformatics software on OSX, but often it requires extra work. If you start using linux now it will pay off in the long run. You can install linux dual-boot on your Macbook Pro.

[kp5091]

Hi Richard, Thank you very much for helping me to install all softwares. I am now following your instruction for installation of Usearch and Bowtie2 from your blog. However, I get some errors like follows:

ksu-wireless-232-89:~ Andy$ echo $PATH
/Users/Andy/.rvm/gems/ruby-2.0.0-p247/bin:/Users/Andy/.rvm/gems/ruby-2.0.0-p247@global/bin:/Users/Andy/.rvm/rubies/ruby-2.0.0-p247/bin:/Users/Andy/.rvm/bin:/usr/bin:/bin:/usr/sbin:/sbin:/usr/local/bin:/opt/X11/bin:/Users/Andy/apps/bin
ksu-wireless-232-89:~ Andy$ export PATH=$PATH:/a/new/path
ksu-wireless-232-89:~ Andy$ mkdir ~/apps
ksu-wireless-232-89:~ Andy$ cd ~/apps/
ksu-wireless-232-89:apps Andy$ mkdir bin
ksu-wireless-232-89:apps Andy$ echo "export PATH=$PATH:~/apps/bin" >> ~/.bashrc
ksu-wireless-232-89:apps Andy$ source ~/.bashrc
ksu-wireless-232-89:apps Andy$ ls
bin
ksu-wireless-232-89:apps Andy$ cd ~/apps/
ksu-wireless-232-89:apps Andy$ curl -o usearch http://drive5.com/cgi-bin/upload3.py...02208022513862
######################################################################## 100.0%
ksu-wireless-232-89:apps Andy$ chmod +x usearch
ksu-wireless-232-89:apps Andy$ ln -s usearch ~/apps/bin/usearch
ksu-wireless-232-89:apps Andy$ usearch
-bash: usearch: command not found
ksu-wireless-232-89:apps Andy$ cd ~/apps/
ksu-wireless-232-89:apps Andy$ curl -o http://sourceforge.net/projects/bowt...4.zip/download
curl: no URL specified!
curl: try 'curl --help' or 'curl --manual' for more information
ksu-wireless-232-89:apps Andy$ curl -o bowtie2.zip http://sourceforge.net/projects/bowt...4.zip/download
######################################################################## 100.0%
ksu-wireless-232-89:apps Andy$ unzip bowtie2.zip
Archive: bowtie2.zip
End-of-central-directory signature not found. Either this file is not
a zipfile, or it constitutes one disk of a multi-part archive. In the
latter case the central directory and zipfile comment will be found on
the last disk(s) of this archive.
unzip: cannot find zipfile directory in one of bowtie2.zip or
bowtie2.zip.zip, and cannot find bowtie2.zip.ZIP, period.

I think that my PATH looks wrong? or weird? because it looks different with the example on your blog. What do you think about it? Could you help me how to figure out and run well them on my computer?

Thank you very much in advance.

[Blahah404]

Hmm, my fault for various typos in some commands. Sorry about that. I've corrected them on the blog post. To fix your situation, run the following in the terminal:

The first thing to do is to remove the last line we added to your ~/.bashrc file. I don't have a mac here, but this should do it:

# check what the last line is
tail -2 ~/.bashrc
# delete the last line we added to .bashrc (don't do this more than once!)
sed -i '' -e '$ d' ~/.bashrc
# check that it's gone
tail -2 ~/.bashrc

You might see an error, but it should still work - you can tell because the last line (output of tail) should have changed after the sed command.

Now we can go through repairing the rest...

# add to the PATH
echo "export PATH=\$PATH:~/apps/bin" >> ~/.bashrc
# load the new PATH
source ~/.bashrc
# go to ~/apps
cd ~/apps/
# redownload bowtie
curl -L -o bowtie2.zip http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.1.0/bowtie2-2.1.0-macos-x86_64.zip/download
# extract it
unzip bowtie2.zip
# add symlinks to ~/apps/bin
cd ~/apps/bin
ln -s ~/apps/bowtie2-2.1.0/bowtie2* .
# fix the symlink to usearch
ln -sf ~/apps/usearch .

Now you should be able to see usearch and bowtie2 symlinked in your ~/apps/bin directory. To check do:

ls ~/apps/bin

And then check you've got usearch and bowtie2 in your path by running:

usearch

and

bowtie2

did that work?

[kp5091]

I got another error with commend line you suggested.

ksu-wireless-232-89:apps Andy$ sed -i '$ d' ~/.bashrc
sed: 1: "/Users/Andy/.bashrc": invalid command code A
ksu-wireless-232-89:apps Andy$ cd ~/apps/
ksu-wireless-232-89:apps Andy$ sed -i '$ d' ~/.bashrc
sed: 1: "/Users/Andy/.bashrc": invalid command code A
ksu-wireless-232-89:apps Andy$ cd ..
ksu-wireless-232-89:~ Andy$ sed -i '$ d' ~/.bashrc
sed: 1: "/Users/Andy/.bashrc": invalid command code A

I do not know what does it mean.

[Blahah404]

hmm, sed is different on the mac. I've updated the previous post with a command that should work, please try again. Even if you get an error, don't run the sed command more than once without checking what it did.

[kp5091]

Richard,

Thank you so much. Finally, I installed both Usearch and Bowtie2 on my mac.
I am now moving on installation of eXpress. Your hands are magic for me.

Andy

[kp5091]

Richard,

I need your help again.
I tried to run transrate and got an error message as follows:

ksu-wireless-235-108:ESTs Andy$ transrate --assembly Aa_Trinity_copy.fasta --reference A_americanum.sequence.fasta --threads 4


Analysing assembly: Aa_Trinity_copy.fasta

calculating contig stats...
...done in 45.990717 seconds

no reads provided, skipping read diagnostics

calculating comparative metrics...
/Users/Andy/.rvm/gems/ruby-2.0.0-p247/gems/transrate-0.0.10/lib/transrate/usearch.rb:12:in `initialize': usearch not found in path. Please ensure usearch is installed and aliased as 'usearch' in your path. (RuntimeError)
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/gems/transrate-0.0.10/lib/transrate/comparative_metrics.rb:13:in `new'
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/gems/transrate-0.0.10/lib/transrate/comparative_metrics.rb:13:in `initialize'
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/gems/transrate-0.0.10/bin/transrate:71:in `new'
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/gems/transrate-0.0.10/bin/transrate:71:in `<top (required)>'
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/bin/transrate:23:in `load'
from /Users/Andy/.rvm/gems/ruby-2.0.0-p247/bin/transrate:23:in `<main>'

Actually,
I installed all software that I need to run translate, but I do not know what is wrong with it.

Could you please help me again?