Level: beginner

This tutorial will show you how to assemble contigs from chromatogram/fasta/seq files with DNA Sequence Assembler.

During the assembly the following steps are automatically performed by DNA Sequence Assembler:

• trimming of the low quality bases at the end of the chromatograms
• reverse-complement of input files (if necessary)
• contig ambiguity correction
• vector removal using primer sequences (if option is checked)

Step 1. Choosing the task you want to perform

Run DNA Baser by clicking its icon on your desktop. The Tasks window should be open by default. Tasks is the central point from you access all DNA Baser functions.

We want to assemble some files so we will click the 'Single contig assembly':

This will open the Project Manager. Project Manager is the window from which you choose the input files, assembly parameters and start the task.

Step 2. Importing the input files

In Project Manager, use the Sample Explorer to navigate to the folder where your samples are located. For your convenience we delivered two demo SCF chromatograms (shown in the image below) in DNA Baser package, a reference file and some vectors.

DNA Baser will assemble ALL files in the Job List. Therefore, we need to add SequenceA.scf and SequenceB.scf into the Job List. There are multiple ways to do this:

• use the single arrow button to add the file(s) selected in Sample Explorer to the Job List.
• use the double arrow button to add all files in Sample Explorer to the Job List.
• use the mouse to drag and drop files from Sample Explorer into the Job List.

If you want to remove files from the Job List, use the button or press the Delete key.

Step 3. Change the assembly parameters (optional)

Go to the Sequence processing option tab and change the parameters for:

• Assembler engine
• Trimming engine (for trimming the untrusted regions of the chromatograms)
• Sequencing error correction

Note: It is necessary to change these parameters ONLY if you have low quality samples and DNA Baser cannot assemble them. Otherwise use the default settings.

Other optional steps:

• In the Vector removal tab select the primers you want to use for the removal of contaminant vector sequences. For your convenience, there are several primer sequences already enter into the program
• In the 'Assemble to reference' enter a reference file. (This is done in a separate tutorial)

Step 4. Project name (optional)

In the 'Project name' collapsible panel (see image above), choose a name for the current project so you can easily identify it later.

This step is optional as DNA Baser will automatically choose a name for your project if 'Auto generate project name' is checked:

Step 5. Assembling the samples

To start the assembly process, press the Start sequence assembly button. During assembly, DNA Sequence Assembler will:

• automatically detect and trim low quality regions of your samples
• automatically trim vector sequences
• automatically correct the ambiguities in your contig
• automatically save the project and the contig to disk

Step 6. Inspecting the assembly

DNA Baser should assemble the demo SCF files provided in less than 0.1 seconds. When done, it should display the assembly in a new Assembly window.

In this window, you will see the alignment of the input sequences, the corresponding chromatograms, the contig and the contig map. You can also edit the contig:

Step 7. Analyzing the log

Upon completion the program will automatically switch to the Log tab. The Log shows:

• Information about the quality of the input files
• Total number of successfully assembled input files
• Information about the unassembled samples (and reason why)
• Number of bases left after automatic end trimming
• Parameters used to assemble the samples
• Contig data:
  • Total number of contigs
  • Contig length before/after vector removal
  • Number contig mismatches/ambiguities
  • Mismatch ratio
• Possible errors and warning (The errors are shown in red color; warnings in orange)

Video tutorial

Things you may want to know:

• The process is identical if you want to assemble other types of samples (ABI, FASTA, SEQ, etc).
• Almost everything is configurable in DNA Baser (what to save, which windows to automatically open, chromatogram color, etc). Don't be afraid to experiment.
• The program will show an explicit warning if you try to assemble less than two files.
• DNA Baser will automatically remove invalid samples from the Job List.
• Different file types (SCF, ABI, FASTA, SEQ, etc) can be mixed (assembled) together.
• When you are in doubt, check the Log. DNA Baser outputs valuable information about the assembly process. Hint: set the log to 'Verbose' mode to see more info.
• You don't have to manually save the contig to disk. It will be saved automatically be DNA Baser. The log and the project CAN be also automatically saved to disk.

The next tutorial explains how to:

• edit the input samples
• correct the ambiguities
• remove vectors
• save the assembled contig.

If you have any questions, don't hesitate to contact us.