[GenoMax]

https://www.biostars.org/p/12475/

Looks like one of your files has good # of reads mapped where as the other one has far fewer. You should be able to look at both of them in IGV at the same time (if you are finally able to figure out how to use IGV).

[GenoMax]

What does it mean

https://www.biostars.org/p/12475/

[Milestailsprowe]

Oh. I have one file that I got to work in goldenhelix. I just compare it to the the one I showed you and there is a big difference in the %. What does that mean

[Milestailsprowe]

Can you run the samtools flagstat/idxstats commands on your BAM file and post the output? That would give us an idea of what the status is as far as alignments are concerned.

Did you try my suggestion with the iGenomes files/IGV?

What does it mean

[GenoMax]

Can you run the samtools flagstat/idxstats commands on your BAM file and post the output? That would give us an idea of what the status is as far as alignments are concerned.

Did you try my suggestion with the iGenomes files/IGV?

[Milestailsprowe]

@GenoMax
Your comment got me thinking, and I've finally found out how come IGV is the only program that can handle smoothly both UCSC and Ensembl chromosome nomenclature. IGV uses a chromosome name alias file.
Simple, but so useful.

"Note: Certain well-known aliases are built into IGV and do not require an alias file. These include mappings that involve adding or removing the prefix "chr" to the name, for example 1 -> chr1 and chr1 -> 1. Also, NCBI identifiers that start with "gi|" and follow the pattern illustrated in the example above are automatically mapped. "

https://www.broadinstitute.org/igv/LoadData

@Milestailsprowe
It really shouldn't be necessary to load your own genome fasta file, or your own GTF file. If you absolutely insist on doing it, the Gene file would be the GTF file.

I'm wondering if you aren't using an ancient version of IGV. The chromosome alias file only appeared in IGV 1.53, which would explain why you can't view your data, if the chromosome nomenclature is different. @Genomax's solution to load your own genome file would then be correct, although a better solution would be to upgrade IGV.

Check which version of IGV you are using. If it's a prehistoric version, just upgrade to 2.3 before doing any more troubleshooting. Then select mm10, reload the BAM file, and view a region that you know has high coverage.

I don't recognize the interface you show in your screenshot in recent versions of IGV, and I believe the interface should be the same across all operating systems. I've only seen this interface in very old versions of IGV.

I could be completely wrong, of course. It wouldn't be the first time.

I'm usuing version 2.3.14 (16)

Can you please give me a run down on how you would do it from start the finish. NOthing is showing up

Where would you download your orginal file? Please. dont understand how I can have file multiple gigs big and no data show up

[GenoMax]

Where do I put the .gtf file?

Thank you for all your help. I'm a graduate student trying to figure this out and my professor never has time sadly

Point to the GTF file in the option box that says "Gene" file.

You can also read in the GTF file along with your BAM later on.

[blancha]

@GenoMax
Your comment got me thinking, and I've finally found out how come IGV is the only program that can handle smoothly both UCSC and Ensembl chromosome nomenclature. IGV uses a chromosome name alias file.
Simple, but so useful.

"Note: Certain well-known aliases are built into IGV and do not require an alias file. These include mappings that involve adding or removing the prefix "chr" to the name, for example 1 -> chr1 and chr1 -> 1. Also, NCBI identifiers that start with "gi|" and follow the pattern illustrated in the example above are automatically mapped. "

https://www.broadinstitute.org/igv/LoadData

@Milestailsprowe
It really shouldn't be necessary to load your own genome fasta file, or your own GTF file. If you absolutely insist on doing it, the Gene file would be the GTF file.

I'm wondering if you aren't using an ancient version of IGV. The chromosome alias file only appeared in IGV 1.53, which would explain why you can't view your data, if the chromosome nomenclature is different. @Genomax's solution to load your own genome file would then be correct, although a better solution would be to upgrade IGV.

Check which version of IGV you are using. If it's a prehistoric version, just upgrade to 2.3 before doing any more troubleshooting. Then select mm10, reload the BAM file, and view a region that you know has high coverage.

I don't recognize the interface you show in your screenshot in recent versions of IGV, and I believe the interface should be the same across all operating systems. I've only seen this interface in very old versions of IGV.

I could be completely wrong, of course. It wouldn't be the first time.

[Milestailsprowe]

In IGV, just pick the mm10 genome instead.
It's the same genome as GRCm38.
I think you'll have to reload the BAM file after you've selected the correct genome.

Then, zoom into a location where you know you will have coverage.
For RNA-Seq for example, you could pick a housekeeping gene, like GAPDH.
Just type GAPDH in the search box, and click on the Go button.

Didnt work

@blancha: mm10 may not work if the one included in IGV is UCSC version which has the "chr" in front of all chromosome numbers.

@Milestailsprowe: If above does not work, create a new "genome" by pointing to the iGenomes (/path_to/WholeGenomesFasta/genome.fa file) and use the corresponding GTF file from (/path_to/Annotations/Genes/genes.gtf). Open your BAM file in IGV.

Where do I put the .gtf file?

Thank you for all your help. I'm a graduate student trying to figure this out and my professor never has time sadly

[blancha]

@GenoMax
IGV is just about the only software program that I know of smart enough not to care about the presence or absence of "chr" in the name of the chromosome, at least for the human and mice genomes. I would congratulate the IGV programmer who programmed IGV not to care whether the "chr" is there or not.

Apparently, the human race is smart enough to sequence the human genome, not smart enough to decide whether chromosome 1 should be written "chr1" or "1".

[GenoMax]

@blancha: mm10 may not work if the one included in IGV is UCSC version which has the "chr" in front of all chromosome numbers.

@Milestailsprowe: If above does not work, create a new "genome" by pointing to the iGenomes (/path_to/WholeGenomesFasta/genome.fa file) and use the corresponding GTF file from (/path_to/Annotations/Genes/genes.gtf). Open your BAM file in IGV.

[blancha]

In IGV, just pick the mm10 genome instead.
It's the same genome as GRCm38.
I think you'll have to reload the BAM file after you've selected the correct genome.

Then, zoom into a location where you know you will have coverage.
For RNA-Seq for example, you could pick a housekeeping gene, like GAPDH.
Just type GAPDH in the search box, and click on the Go button.

[Milestailsprowe]

Which version did you download from iGenomes? This looks like NCBI or Ensembl since UCSC versions have the word chr in front of the chromosome number. This is certainly not the 129S1 mouse genome as you had selected in the screenshot above.

I am going to suggest that you use the sequence and the annotation in your iGenomes download in IGV so everything matches and you can display the data. See "Loading a genome" section.

I downloaded the NCBI GRCm38 version. Should I use something else? Which one would you use?

[GenoMax]

Which version did you download from iGenomes? This looks like NCBI or Ensembl since UCSC versions have the word chr in front of the chromosome number. This is certainly not the 129S1 mouse genome as you had selected in the screenshot above.

I am going to suggest that you use the sequence and the annotation in your iGenomes download in IGV so everything matches and you can display the data. See "Loading a genome" section.

[Milestailsprowe]

My guess is that your BAM file has chromosome names that do not match what is provided by IGV in terms of the reference (e.g. chr2 vs 2). That is assuming you have selected the correct reference (I see Mouse 129S1 etc in the screenshot above).

Where did you get your reference genome from? If you are using a non-standard genome then you can load your own reference sequence in and use it to display data against.

Can you post the header from your bam?

$ samtools view -H your_bam

Thanks for the help. I got the reference one from igenome. Trying to get this mouse data to show.