Hello, I am new to sequencing and this line of work.
For the past several weeks I have been using Geneious, and have collected several sequences that I believe to be a certain protein group. I now want to take those sequences and compare them to my genome to see where they are and how closely they cluster.
I have never used Integrative Genomics Viewer (IGV) before and have been learning from trial & error/tutorial videos.
I have managed to get my genome into the program and can view it properly. Now I want to take the sequences I have in Geneious and align them to the genome in IGV so I can visually see where they cluster.
When I try to export the sequence from Geneious the only file type that can be read by both programs is .gff (while they can both read FASTA files apparently IGV only uses FASTA files for "defining reference sequences; they cannot be "loaded" from the file menu.") I do not know what a gff file is but when I try loading it in IGV it shows up blank. Per the tutorials the loaded sequences will only show up at 30kb zoom. However, even at that magnification I cannot see the sequence.
In the examples I have seen, it appears I need a BAM file, but I do not know what type of file this is or how to create such a file (where does it originate? what program?).
Again I am very novice to this material so my understanding is a bit limited at this time. Any help is appreciated.
Cheers
Hi, do you have access to a Linux server? there are plenty of options to open both .gff and BAM files and get the seqs you want.
Hi Justin,
How large is your genome and how many chromosomes or contigs does it have? In any case, you should have no trouble visualizing where your gene annotations are in the genome. Could you paste here the first 6 lines of your GFF file? Maybe Geneious didn't generate a correct annotation file.
To be absolutely sure, you could try and search for the genomic coordinates or identifier of your sequences in the IGV search bar. For example, chr1:10000-20000; where chr1 should be the chromosome/scaffold, 10000 the start position and 20000 the end position of your sequence. There should be a clarifying pop-up when you place the cursor on the search bar that tells you how to introduce this information. You can also try to look for an identifier that matches the 9th column of your GFF. With both ways you should zoom in on the genomic position of your sequence, and you could see if the annotation is there or not. If it is not there, you definitely have a problem with your annotation file.
I hope this helps. Don't hesitate to ask if I didn't explain it clearly.
If your purpose is to align reads (from next-generation sequencing experiments) to your genome of interest, then the above comments are exactly what you need.
However, it sounds like you are trying to align multiple homologous sequences (from GenBank, or similar) to each other and then perform a clustering analysis. If this is the case, then IGV probably wouldn't be the best visualization program.
Your workflow would look something like this:
1) save the genome you imported into IGV (or region of interest from the genome) as a fasta file
2) save the other sequences from Geneious as a fasta file
3) combine the fasta files from steps 1 and 2 into a single file
4) submit the file to a sequence alignment algorithm. There are public servers with these algorithms installed. Please make sure that you select a "local" alignment instead of a "global" alignment. Otherwise the output will be hard to interpret because of many gaps.
5) continue on with the clustering analysis.
Hope this helps!
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