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Aligning short reads to a reference sequence (Read 6283 times)
Oct 20th, 2016 at 1:43am

girlsciencefriday   Offline
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I am having trouble aligning more than one short read to a reference sequence. All of my sequences are in FASTA format.

In the multiple alignment editor, I can have one sequence listed and select 'Align sequence to profile with MUSCLE' and the alignment will work perfectly. However, using this method I have been unable to align multiple short sequences to a profile. Instead, the sequences all align to each other.

I have also tried using Tools>NGS Data Analysis>Map reads to reference, but when I select my FASTA files for short reads and reference sequence, I get an error message saying "The short reads cannot be aligned to the reference!"

Any help with this process would be great.
 
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Reply #1 - Oct 20th, 2016 at 3:41pm

Olga Golosova   Offline
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Hello!

Could you please clarify what task you're solving?
What kind of data you have - is it a few sequences that you want to align or huge number of short reads (NGS data)?

For example, if you have Sanger reads and have a reference sequence, you may use "Tools > Sanger data analysis > Reads quality control and alignment" workflow. As input you will need to provide the reference sequence and the list of sequences to align to it (e.g. in FASTA format). The output is the aligned reads with the reference that can be opened in the Alignment Editor.

You might also find useful "Align to this alignment" button on the Alignment Editor toolbar. To use it select a sequence object (or several objects) in the project view and click the button. The sequence(s) will be aligned to a proper location, but the original alignment will not change. Note that this button has also other behavior. If nothing is selected in the project view, the button opens a file dialog where you can select the file with sequences.

If you have high-throughput sequencing data (next generation sequencing), which seems is not the case, you may use, for example, the item you mentioned - "Tools > NGS data analysis > Map reads to reference". In this case you also should have the reference sequence.

In short, it seems you may follow one of the ways:
1) Select the Sanger workflow. Input reference. Input FASTA as reads. Run the workflow. Open the result alignment.
2) Open reference in the Alignment Editor. Click "Align to this alignment" when nothing is selected in the project view. Select the FASTA file in the dialog.
 
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Reply #2 - Oct 27th, 2016 at 12:37am

girlsciencefriday   Offline
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Thank you!! I was working with just a few sequences and your second suggestion worked perfectly!
Cheesy
 
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Reply #3 - Oct 27th, 2016 at 6:26pm

Olga Golosova   Offline
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Great! Smiley Thanks for informing me!
 
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