Align to Reference Sequence

[traini82]

I am assembling genomic resequencing data for a specific gene and would like to align to reference so that I can use a single reference file for all exons and have each assembly (by assay name) be the size of the amplicon rather than the huge open reading frame genomic sequence. I can achieve this by manually trimming each assembly but this is very tedious especially for large genes (ie 30-50 exons). Is this possible?

Thanks

[Peter Richterich]

Each alignment to a reference sequence will cover the entire reference sequence. To generate one alignment for each exon, you would have to create separate reference sequences for each exon. You can include all these references sequences when you make the alignment, and the sequences should automatically align to the correct reference sequence (assuming that each sequence covers only one exon).

[traini82]

Could I make a request to add this to the 'wish list' as such a function would also be extremely useful in cases where we are doing SNP discovery in cell line cDNA. We could use genomic reference which has known SNPs annotated in it. It would be the reverse of Sequenchers ability to insert large gaps when assembling gene sequence to large genomic reference and preclude us having to scan through the unaligned reference's 'known SNPs' and focus on the aligned areas (experimental) polymorphisms.

Thanks

[Peter Richterich]

We have added better support for multi-exon sequences to our list of requested features. Unfortunately, the request came to late to make it into the upcoming next major release, but it has been marked as high priority for the version after the next. Would it be ok to contact you with additional questions about this in the future (although it seems that our last 2 emails have not reached you, probably because they were misclassified as junk)?

[traini82]

Sorry to hear that the exon feature will not appear in the next build but at least its on the list. It would certainly be ok to contact me with questions about this and any other proposed feature. I'm not sure why emails were not received if sent to the address provided at registration. I haven't noticed any codoncode addresses in my quarantine summary (I did get the registration acknowledgement.)

Thanks

[traini82]

I believe the new Reference Shortening feature in v 3.5 which allows automatic clipping of very long reference sequences to the region you want to look at addresses the multi-exon support previously discussed in this thread? I'd like to try it but unfortunately there are no notes where to find it or how to use it.

[Peter Richterich]

How uncovered reference sequences are shortened is determined by the "Alignment" preferences; the option at the bottom allows you to set whether or not uncovered references should be clipped (and, when clipping, how many extra bases to leave). You can find a Flash tutorial at www.codoncode.com/aligner/tutorials/Auto_Shorten_Ref_Seq.html.

Note that you will need to generate the alignments separately for each exon. The best way of doing this is by using "Align in Groups" (Contig menu => Advanced Alignments). There is a Flash tutorial for assembling in groups at www.codoncode.com/aligner/tutorials/ByName.html. Align in groups follows the same logic when you have a reference sequence.

[traini82]

Thanks for the links, is there a way to use "Align in Groups" to assemble to a reference sequence (i.e. Sequencher's "To Reference by Name")

[Peter Richterich]

is there a way to use "Align in Groups" to assemble to a reference sequence (i.e. Sequencher's "To Reference by Name")

Yes, there is a menu option called "Align in Groups" in the "Advanced Alignments" sub-menu in the "Contig" menu.

You can find detailed information about how to use this option in the online help as follows:

1. In CodonCode Aligner, go to the "Help" menu
2. Select "Aligner Help..." to display the online help
3. Click on the little book icon near the top left to display the index on the left side
4. Click on the triangle next to "Assembly and Alignment" to expand the topic, then do the same for "Align to Reference", then "Advanced Alignment Options"
5. Click on "Align in Groups"