Dear colleagues,
I want to sequence some genes from different mammals species. However, the major part of them has no reference sequence. What is the best strategy to do it? Is there any free available software that I can use?
Thanks in advance for your help!
Best,
David
I have had to do this myself and it's a pain. The best thing you can do is search databases (NCBI, Ensembl) for sequences of the closest relatives you can find. Put them all together in FASTA format in a .txt document and do a multiple alignment using Clustal Omega. It's best to do these alignments using mRNA/cDNA transcripts or complete coding sequences (cds) because introns will not align well. However, you will need to design primers within exons (not across junctions) if you want to sequence DNA. I do this by using Ensembl to figure out where exons are and using the longest exons as the input to primer design programs (like Primer3). Of course, if you can't get a section long enough for your purposes you will need to use Ensembl to look at how far apart the exons are and choose a pair that give good amplicon length, then design primers in each exon individually. I'd recommend excluding sections of the exon that are not well conserved according to your Clustal alignment.
I have had to do this myself and it's a pain. The best thing you can do is search databases (NCBI, Ensembl) for sequences of the closest relatives you can find. Put them all together in FASTA format in a .txt document and do a multiple alignment using Clustal Omega. It's best to do these alignments using mRNA/cDNA transcripts or complete coding sequences (cds) because introns will not align well. However, you will need to design primers within exons (not across junctions) if you want to sequence DNA. I do this by using Ensembl to figure out where exons are and using the longest exons as the input to primer design programs (like Primer3). Of course, if you can't get a section long enough for your purposes you will need to use Ensembl to look at how far apart the exons are and choose a pair that give good amplicon length, then design primers in each exon individually. I'd recommend excluding sections of the exon that are not well conserved according to your Clustal alignment.
The use of molecular markers such as mitochondrial genes, in special COI has been extensively used to species delimitation in different orders of animals including mammalians. However another regions can also be used. In order to define which is the best for your research, you can go Taxonomy data base in NCBI, and look the genes or regions used for mammalia taxonomy. I suggest to use the most frecuently used because it will be help you in the species delimitation, since the aligment will be more easily,
Thank all of you for your answers to my question. I can understand the idea. However, and as Beatrice said it will be more complicated in practice for sure. I will try it and if I had another question I will ask again.
Thanks for your support,
Best
Hi David,
seems like you need to design degenerate primers, and as mentioned above it may not be very easy.
In a nutshell, you need to prepare a DNA or protein alignment using the sequences from the closely related species, then find the most conserved part in it and design a set of primers, which would account for all possible nucleotide combinations (that is a mix of primers with variable nucleotides in some positions). There are some programs that allow to do that, for example http://acgt.cs.tau.ac.il/hyden/HYDEN.htm and http://blocks.fhcrc.org/codehop.html
Here is a good primer on how to design degenerate primers:
http://bitesizebio.com/18992/a-primer-for-designing-degenerate-primers/
Good luck!
Olga
Hi David,
I spent years as a postdoc designing primers for to amplify regions of interest from the dog before the sequence was published. I used the known sequences from mouse and human to choose areas of high homology in exons. I'm not sure if this will help you, but here is a link to the paper we published 10 years ago (free access). There are likely newer methods and certainly newer software, but perhaps it will provide some insight that might still be relevant. Good luck with your research.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635982/
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