This page provides a quick introduction to installing scaffolder and creating a simple scaffold from three nucleotide sequences. Scaffolder has been tested on Debian and OS X but there may differences on Windows. If you have any questions or comments feel free to ask on the scaffolder mailing list.
Scaffolder requires both Ruby and RubyGems to run. These are usually already installed or can be installed easy on Mac OS X and Linux distributions. The software can be tested by running the following commands on the console or terminal (excluding the '$' sign):
$ ruby -v $ gem -v
If both of these return without error, scaffolder can be installed via RubyGems using the following command:
$ gem install scaffolder scaffolder-tools
The install can be tests by running the following command. This should print the list of available scaffolder options.
If this fails or returns an error the RubyGems executable directory may not be in your shell path. The location of this can found using the following command, the result of which should be added to the $PATH variable in your shell login script. Examples of updating the shell path are available for OS X and for Linux
# Add the output of this your $PATH $ gem env | grep "EXECUTABLE DIRECTORY" | cut -d : -f 2
Creating a simple scaffold
To begin creating a scaffold, create a text file named 'sequences.fna' containing the following nucleotide sequences. These will be our example sequences which we wish to build a scaffold with. The following steps will progress through creating a simple scaffold.
>sequence1 AAAAAAAAAAAA >sequence2 TTTTTTTTTTTT >sequence3 GGGGG
Create the file 'scaffold.yml' and add the content below. This is the scaffold file specifying how our nucleotide sequences should be joined together.
--- - sequence: source: 'sequence1'
The scaffold is written in the YAML format. YAML is data serialisation format which is useful to pass information between computer software but which also can be easily edited by hand. YAML formatted documents should begin with three dashes ('---'). The scaffold file is represented as a list of sequence entries. Using the YAML format each entry is defined by starting with a dash ('-') then indented by two spaces for the remaining lines of the entry.
As you can see in the code above this scaffold file has only one entry. The 'sequence' tag specifies that this entry is a sequence. The 'source' tag identifies the name of the sequence to use. The source tag should match the first space delimited word in the corresponding sequence fasta header.
The sequence for this simple scaffold is produced by running the scaffolder sequence command giving the locations of the two files we have just created.
$ scaffolder sequence scaffold.yml sequences.fna
This returns the nucleotide sequence of sequence1. This is as expected as the scaffold only contains one sequence entry.
Adding additional sequences to the scaffold
A scaffold with only a single sequence is rather simple. Update the scaffold file so that it now resembles the text below. Here we have added a second sequence from the fasta file and an unknown region between these two sequences.
--- - sequence: source: 'sequence1' - unresolved: length: 5 - sequence: source: 'sequence2'
Running the scaffolder command again will generate following super sequence. This contains the two sequences joined by the 5bp region of 'N' characters we specified. These unresolved regions can be used for joining contigs by approximate distances.
Replacing regions of sequence in the scaffold
Imagine there is a region in sequence1 which we wish to replace. For example the start of region on is poor quality or it is an already scaffolded set of contigs containing gaps. Performing PCR would determine the correct sequence in this area but we need a way to update sequence1 in the scaffold. We can do this by updating the scaffold file as follows:
--- - sequence: source: 'sequence1' inserts: - source: 'sequence3' open: 3 close: 5 - unresolved: length: 5 - sequence: source: 'sequence2'
Here we have added an insert to sequence1 using the "inserts" tag. This replaces the region in the range 3...5 (specified by the open and close coordinates) with sequence3. Running the scaffolder command again we can see that this is now the case where four guanine bases have been inserted
This is a brief tutorial for creating a simple scaffold. More complex scaffolds can be created by adding further sequences and inserts to the scaffold. More details about the scaffolder format can be found at the scaffolder API and the scaffolder-format man page. Details on the available scaffolder commands can be found on the man pages.