view alignment errors

[Yan Yan]

Hi colleague, 

I am trying to use IGV to view my sequencing alignment. 

The sample is one of metagenome sample contained all the possible concatenated markers of Prevatell copri. I converted the sample .sam file to .bam file and got the sorted bam and index files by igvtools. See this sample example: https://www.dropbox.com/sh/gjphcom7uyqjqvi/AABFNn0iAopuLyvghL4GcLNBa?dl=0 

I have downloaded reference genome of P.copri from NCBI: https://www.ncbi.nlm.nih.gov/assembly/GCF_000157935.1 

I load genome from Genomes dropdown. However, when I load sample files, it tells https://www.dropbox.com/s/vv543jomlals0lp/Screen%20Shot%202021-02-21%20at%204.32.32%20PM.png?dl=0 

I am not sure where went wrong. I would appreciate any helps. 

Thank 

Yan

[Helga Thorvaldsdottir]

Hi Yan,

I took a look at the files you posted in that dropbox folder and your bam file does not appear to be aligned to that reference.

The sequence names in the. bam file are like: 165179__A0A174IHQ2__PREVCOP_05478

The sequence names in the .fna file are like: NZ_GG703857.1

Helga

[Yan Yan]

Hi Helga,

Thanks for your checking.

Let me introduce a bit about the sample.bam. You may know strainphlan3 from Huttenhower group from Broad. I am currently metagenomic samples from strainphlan3. And the sample.bam contained all the concatenated markers of Prevatella copri that I extracted from metaphlan3 intermedit output .sam file and converted to the bam file as you see in dropbox. I got your point and I found out the sequences/genome from P.copri, which I uploaded here:  https://www.dropbox.com/s/z3wti7ipazq3jbt/s__Prevotella_copri.fna?dl=0 my question is that there are far less marker genes in the bam file, but more in the P.copri sequences/genome, do I need to subtract to the ones that only in the bam file? I tried to see in IGV, it seems working. Thank you!

Cheers,

Yan

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[Helga Thorvaldsdottir]

IGV needs to know where to place the read alignments agains the reference genome. So if the BAM file refers to a sequence/chromosome that does not exist in the reference genome, the alignment can't be displayed. However, there can sequences/chromosomes in the reference genome that nothing is mapped to. You don't need to remove sequences/chromosomes from the reference genome just because nothing is mapped to them.

Helga

[Yan Yan]

Thank you. That makes sense to me. Since I see some of the reads nothing map, but some are mapped to the reference genome. 

Cheers,

Yan 

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[Vivek Keshri]

Hello, 

I am new to IGV. I want to check the query sequence alignment on the genome (for the location of mapping/aligned regions in the genome). 

I uploaded the Genome (.fasta file) on IGV and now trying to upload my query sequence (*fasta file), but it is not working. Kindly suggest to me how to upload the query/ alignment sequence??  If it supports only the BAM file then please let me know how I can convert my alignment file (.fasta) to the BAM? 

Thanks in advance, Looking forward to your favourable response. 

Vivek

[Helga Thorvaldsdottir]

Hi,

You can't just convert a fasta file to the aligned bam file format that IGV needs.  You need to use an aligner tool to align the sequences to a reference sequence.  Or perhaps you're looking for a different type of viewer? - perhaps a multiple sequence alignment viewer? You'll find a few via a google search.

Helga

[Helga Thorvaldsdottir]

Vivek, this is not the right forum for advice on that type of question.  Perhaps consult with someone where you got the files from or with colleagues doing similar work. There are also more appropriate forums, such as biostars.org and bioinformatics.stackexchange.com 

Helga 

On Thursday, April 8, 2021 at 1:12:00 AM UTC-4 Vivek Keshri wrote:

Thank you Helga. Please suggest to me any aligner tool whether I can get BAM file in output (for IGV input). I have some query sequences and a reference genome sequence in Fasta file format. I am trying to align the query sequences in order to find the location of all sequences in the reference genome. 

Thanks 

Vivek

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