NEw to Chip-seq and have .bam/.sam/.bam.bai files... then what?

[NGS newbie]

Hello everyone!

I have recently received chunks of data files (.bam/.bam.bai/.sam/fa, etc). After panicking for a moment, I started the "genomeview" program online. I was able to upload my data (.bam.bai file), then an error message popped up. Then I uploaded the .fa file for rat chromosome sequence (rn4). I am trying to annotate this data and I am at a loss. I think the problem is that my core personnel aligned the data to something different than what the Avadis NGS or the UCSC genome browser uses, so none of these programs will give me names of genes to which my samples aligned. HELP!!!

[NGS newbie]

I guess what I am really asking is, how do I annotate my rat ref seq data?

[Mamoon Rashid]

Hi,
I first load fasta file (to which reads have been mapped) to any genome viewer (i use Artemis), then i load .bam file. Please ensure that you must have ".bam.bai" file in the same directory where you place ".bam" file. Please let me know if this solves your problem.
Thanks

[NGS newbie]

Thank you, but my problem isn't loading the sequence file with the fast file, my problem is annotation. I have everything loaded on the genomeview program, my bam.bai file and the fast file, but other than the actual nucleotide sequence at a given position on a chromosome, I have no idea what genes are present. also, the chromosome info is presented as gi|xxxxxx|ref|NC_xxxxxx. I just dont think I can analyze data not knowing what is where, unless i have eternity to sift through everything o the genome one gene by one gene.

Thank you so much for your help though!

[Mamoon Rashid]

Annotation

let me ask few questions-
1. Did you map short reads to reference? Which genome you are trying to annotate? OR are you trying to annotate the reference genome with short read alignment, like we do with RNA-seq data?
2. Which genome viewer are you using?

Thanks

[NGS newbie]

answer

Hello!

I'm using a freeware called "genomeview." I am trying to map a chip-seq short read data generated from illumina ga platform. Single, not paired. I had the core personnel align my reads to rat ref seq rn4 (RGSC).

I also tried using avadis ngs and when I upload my bam or sam file to their preloaded ref seq annotated sequences, none of my reads map. It is bizaar as I have over 20 million reads and I don't see any peak. Sigh.

[Simon Anders]

Annotation is typically provided in GTF files. Get a GTF file for the same built of the reference assembly as the one your core facility aligned your read against, and load it in the browser, alongside your reads. You may need to replace the chromosome name in case your GTF file does not use the same long RefSeq/UCSC ID format as your FASTA files did.

[NGS newbie]

Ill definitely try that 1st thing in the morning. Where do I get gft files from? I think I've seen them in FTP files from rgsc or pubmed site but not sure if there is one file for all the chromosomes or one file per chr.

Thank you so much for your help!

[NGS newbie]

Oh also, how do I rename chr?

[Strand SI]

Dear NGS Newbie,

Chances are that the chromosome name used in the imported sample and the chromosome name used in the Avadis NGS rn4 build are not matching. Typically it is a simple issue of adding aliases to chromosome names in the Annotation Manager in Avadis NGS to get things working. Please contact our support team online or write an email to [email protected] and we will assist you with this.

The Avadis NGS Team
www.avadis-ngs.com

[Mamoon Rashid]

conflict of IDs

Yes, I have also noticed recently that your reference fasta file upon which you try to load the BAM file and the reference used in mapping reads must be same. I mapped short reads against a bacterial genome ( having two chromosomes in the same file) with BWA and samtools. When i loaded the BAM onto the same reference in Artemis nothing was matching. Then after a day troubleshoot i edited the original reference fasta file and formatted the header as mentioned in the BAM file, then everything go smooth.
You can see the header in BAM file by-
samtools view -H <any>.bam
These headers must be equal to those present in the reference file.
Thats what i learnt.

[Strand SI]

add differing chromosome names as aliases

Oh also, how do I rename chr?

There's no need to rename the chromosomes in your file. In Avadis NGS, you can look up what the actual chromosome names in your file are and then add those chromosome names as aliases in the Annotations Manager. We've put together a short video that shows you how to do that:

www.avadis-ngs.com/support/video-tutorials/chromosome

The Avadis NGS team