Samtools: Problem Not Recognizing Sorted BAM File & Output Missing Contigs

[IllumMiseq]

Hello!

I converted my SAM file to BAM using this command:

samtools view -S -b .SAM > M6X2D.BAM

I need to calculate genome coverage, so I'm using this command:

samtools depth .BAM > .txt

But I got this error:

[bam_pileup_core] the input is not sorted (chromosomes out of order)
[bam_plp_destroy] memory leak: 1. Continue anyway.

I did a google search and saw the following command could be used to sort my BAM file:

samtools sort -n .BAM

But it doesn't help. I would appreciate any advice and guidance on what I can do to get the depth command to work.

I did get the depth command to work for one genome, but only 2 out of 46 contigs made it to the .txt file.

Thank you!

[GenoMax]

What version of samtools are you using? You should use the latest (v.1.5) if possible.

Sort your BAM file this way "samtools sort -o sorted.bam original.bam". (-n will name sort based on reads which is not what you want here).

[IllumMiseq]

Hi! I'm using samtools 1.6.

I tried to sort the BAM file as suggested using "samtools sort -o sorted.bam original.bam", but I got this here:

[E::hts_open] fail to open file 'Sorted.BAM'
[bam_sort_core] fail to open file Sorted.BAM

I tried to switch around the sorted and the original bam components like this:

samtools sort original.BAM -o Sorted.BAM

and my computer screen went crazy.

[GenoMax]

You need to use real file names (with full paths, if needed). So you would do "samtools sort -o M6X2D_sorted.BAM M6X2D.BAM" (if that is a real file name).

[IllumMiseq]

Correct. That's what I did. I had the path set for my files. I did tab complete just to be sure. It just says:

open: No such file or directory
[bam_sort_core] fail to open file M6X2_Sorted.BAM

My exact command was:

samtools sort -o Strain_Sorted.BAM ../Novels_BAM/Strain.BAM

[GenoMax]

That is odd. Why is the error saying "fail to open file M6X2_Sorted.BAM" when your input file is called "Strain.BAM?

[IllumMiseq]

I have multiple single genomes that I'm working on. I just replaced M6X2 with "Strain" to put a generic name to my files while we are chatting.

samtools sort -o M6X2_Sorted.BAM ../Novels_BAM/M6X2.BAM

is my literal command.

[GenoMax]

I see. You are trying to write M6X2_Sorted.BAM to the local directory from where you are running sort command and you do not seem to have write permissions there.

So it would be best to either put the sorted file back in the directory where the original BAM is by doing "samtools sort -o ../Novels_BAM/M6X2_Sorted.BAM ../Novels_BAM/M6X2.BAM" or put it in another directory, where you have write permission.