Get single pages from a PDF with pdftk

Another little trick for file manipulation in Linux, which I find useful when making/reading papers, is how to convert a multi-page PDF document into multiple single-page files.

Here is how I do it in Ubuntu, using the lovely program pdftk:

 for i in {1..7}; do echo Working on page $i; pdftk A=INFILE.pdf cat A$i output OUTFILEpage$i.pdf; done

Installing CASAVA/bcl2fastq on Ubuntu

I've been playing around with working some of my own demultiplexing scripts into my current analysis pipeline, so I thought I'd best get to grips with CASAVA, or bcl2fastq, so I can de-demultiplex (multiplex?) my MiSeq data.

The trouble is, CASAVA is not supported for my OS, Ubuntu (for reference, I'm running 13.04). Still, I thought I'd give it a try, can't take too long right? Wrong. Most painful installation ever.

I won't bore you with all the details, but here are the major stops and errors I got along the way, to help people find their way if they find themselves similarly stumped.

I started off trying to install CASAVA v1.8.2, the most up to date version I could find (before I realised that CASAVA has since turned into bcl2fastq).

I tried to build from source as per the instructions, but was unable to make:

make: *** No targets specified and no makefile found. Stop.

Checking out the log, revealed that boost was failing; it couldn't find the make file as the configure hadn't made it.

So, then I tried installing boost via apt, which let configure run fine, but everything ran aground during the make due to incompatibility issues. I should have expected this; the version on boost available through apt was several versions newer than that which comes bundled with CASAVA (1_44) (I know right, the apt version being too new? I didn't see it coming either).

Removing the version of boost I just installed seemed to now allow the bundled version to take over with the make, but now it encountered another issue:

/usr/local/CASAVA_v1.8.2/src/c++/lib/applications/AlignContig.cpp:35:32: fatal error: boost/filesystem.hpp: No such file or directory
compilation terminated.
make[2]: *** [c++/lib/applications/CMakeFiles/casava_applications.dir/AlignContig.cpp.o] Error 1
make[1]: *** [c++/lib/applications/CMakeFiles/casava_applications.dir/all] Error 2
make: *** [all] Error 2

So far so frustrated. Plan B, try and install the rpm version of the more up-to-date bcl2fastq. Now as an Ubuntu user I have no experience with rpm, but let's give it a go.

Rpm couldn't find any dependencies. I mean, any dependencies. At all. Including this:

/bin/sh is needed by bcl2fastq-1.8.4-1.x86_64

I'm not sure about a lot of the packages installed, but I know I've got sh.

OK, let's try something else; googling around the subject suggests that yum is the way to go. I tried this, it still couldn't find any of the required dependencies.

So I tried one last thing, and installed alien, and used that to install the bcl2fastq rpm...

alien -i bcl2fastq-1.8.4-Linux-x86_64.rpm

...and it worked! Huzzah!

Although I really shouldn't have been surprised. The rpm installation even told me I should:

rpm: RPM should not be used directly install RPM packages, use Alien instead!

TL,DR: Trying to install CASAVA/bcl2fastq on Ubuntu? Try using alien to install the rpm!

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Update for Ubuntu 14.04 (Trusty Tahr):


After recently updating to the newest version of Ubuntu, I've tried to follow my own advice regarding the installation of bcl2fastq, only to find that it no longer works (see below for horrendous stream of errors)!


Luckily the problem - that the newer version of Perl used in Trusty isn't compatible with bcl2fastq - had already been identified and solved by the good people over at SeqAnswers (thanks to Tony Brooks and Hiro Mishima).

"my" variable $value masks earlier declaration in same statement at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 760.
syntax error at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 747, near "$variable qw(ELAND_FASTQ_FILES_PER_PROCESS)"
Global symbol "$variable" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 749.
syntax error at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 751, near "$directory qw(ELAND_GENOME)"
Global symbol "$self" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$directory" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$project" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$sample" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$lane" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$barcode" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$reference" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 753.
syntax error at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm line 761, near "}"
/usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment/Config.pm has too many errors.
Compilation failed in require at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment.pm line 61.
BEGIN failed--compilation aborted at /usr/local/lib/bcl2fastq-1.8.4/perl/Casava/Alignment.pm line 61.
Compilation failed in require at /usr/local/bin/configureBclToFastq.pl line 250.
BEGIN failed--compilation aborted at /usr/local/bin/configureBclToFastq.pl line 250.

Installing python dependencies for Decombinator

I've written previously about Decombinator, the TCR repertoire analysis program developed in our laboratory.

Having just formatted my computer and reinstalled all my packages, I've been reminded of what a faff this can be.

This time around I did things a lot more efficiently than I had before. Seeing as how my colleague responsible for updating the current readme is busy writing up his thesis, here's my quick guide to installing all the python modules required by Decombinator in Linux - at least in Ubuntu.

 sudo apt-get install python-numpy python-biopython python-matplotlib python-levenshtein   
 sudo apt-get install python-pip  
 sudo pip install acora  

Most of the modules are available in the repositories, which has the added bonus of filling in all their required dependencies. Acora (the module that enacts the Aho-Corasick finite-state machine) however is not, but is easily installed from the command line using pip.

Easy. Time to get decombining.

August 2016 update:
Decombinator has gone through a number of improvements since writing this post. The current version (and installation details) can be found on the Innate2Adaptive lab's Github repo.