PacBio tools

SMRT-link software server tool

The sequencing PacBio data is normally processed by a sofware-server tool SMRT-link. Which means that a Software to process the PacBio data should be intalled in a server and then processed there using the standars protocols of pacbio.

Here some additional information related to SMART-link:

• All SMRT software versions

• SMRT® Portal Help

• Installing SMRT_youtube videos

• Evolution of sequencing era

• The white paper

• SMRT link without control?

PacBio-Bioconda

If you are familiar with command lines and programing the PacBio company provide a collection of tools to manage the sequencing data. This can be installed in a server or a personal computer using a virtual environment called Bioconda, specifically Miniconda. The collection of tools are called the PacBio Bioconda tools.

Installing Miniconda

It is possible to work with the PacBio data using differents tools installed through Bioconda (virtual environment).

Follow the next steps to install Bioconda and its most useful tools.

1. Download miniconda Choose between miniconca 2 (for using python 2) and miniconca 3 (for using python 3) in the miniconda webpage depending which version you need to work, choose the operative system you are using and donwload it.

To download miniconda 2 in MacOS, you can type the following line in your terminal:

curl -O https://repo.anaconda.com/miniconda/Miniconda2-latest-MacOSX-x86_64.sh

To download miniconda 3 in MacOS, you can type the following line in your terminal:

curl -O https://repo.anaconda.com/miniconda/Miniconda3-latest-MacOSX-x86_64.sh

2. Install miniconda Using the terminal in the directory where miniconda was downloaded, the following line if you want to install miniconda 2 or 3 (just change the name). In this case miniconda 3 is going to be installed:

sh Miniconda3-latest-MacOSX-x86_64.sh

1. Standard directory to install miniconda is in you home directory and a directory called miniconda3 will be created. Don’t forget the location of this folder because this is the route to call the virtual environment.

2. The installation process also ask you if you want to specify a directory. You can create a new one only typing a directory name after the >>>prompt. This is optional and useful when you want to have more tha one virtual environment related to miniconda.

3. Another important question during the installation is if you wish the installer to initialize Miniconda3, wich means that every time you start the terminal, the virtual environment will be activated and the tools installed there. You only need to type ‘yes’ or ‘not’ after the ‘>>>’ prompt.

3. Activate miniconda If you type ‘not’ in the las step, then, when you open the terminal, go to your home directory where miniconda was installed and activate it as the following example (activation for miniconda3):

source home_directory_route/miniconda3/bin/activate

4. Configure miniconda The first step is to have miniconda activated. Then, to configure miniconda installation, type the following lines in this order:

conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

5. Installing tools The more useful tools can be installed using the following line:

conda install -c bioconda blasr pbcore numpy scipy pbbam samtools pbcommand

If you are interested in other tools, check the collections of tools you can install in this link –> PacBio-Bioconda tools Is recomendable to update the tools after installation. To do this type:

conda update tool_name

for invidual tools or:

conda update --all

to update all the tools.

6. Deactivate miniconda To deactivate the miniconda you don’t need to be in the home directory, just type this line and miniconda will be deactivate:

conda deactivate

Some of the more useful tools to consider are:

Tool	Description
bax2bam	Allows to transform files from old chemiestries and formats to the bam file format.
blasr	The official PacBio aligner adapted for long sequencing reads. Although other aligners such as BWA, Segemehl and pbalign were compared, blasr had the best mapping along with pbaling, both aligners found in the pacbio bioconda tools. It was decided to take blasr as the aligner to do the analyses because it is the only one whose result includes the ipd columns needed to be able to detect DNA modifications.
pbmm2	New aligner suggested to be a substitute for blasr. When evaluated, it turned out to be faster in the alignment process, however there is not a big difference in the total number of aligned subreads. The output was not sorted by molecule and has therefore been discarded for the time being.
pbccs	This produce the Circular Consensus Sequencing from all the subreads on each molecule