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Old 04-22-2016, 02:16 AM   #1
erica.n
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Default 8 column output file from SAMtools mpileup

Hi all,

First let me briefly describe what I am trying to do. I have Illumina reads (ezRAD and Illumina MiSeq), from which I want to identify putative neutral and adaptive SNPs. Here's what I have done so far:

- trimmed the reads for adaptors and quality with FASTQC toolkit
- inported the reads into GALAXY
- groomed the reads so that they were converted from phred+64 to phred+33
- mapped these reads onto my reference genome (L. gigantea) with BWA-MEM with default parameters
- cleaned the output BAM files with CleanSAM in SAMtools using the strict option
- filtered the BAM files to remove alignments with MAPQ < 15, using Filter SAM tool under SAMtools in Galaxy
- Merged my BAM files so all of my samples were combined, using merge BAM tool under SAMtools in Galaxy, with default parameters
- performed an Mpileup on the merged BAM file using SAMtools, where I did not perform genotype likelihood computation, with the same reference genome and basic parameters

After performing the Mpileup, I got a pileup output file that looks like this (only showing the 1st two lines):

1 2 3 4 5 6 7 8
LOTGIsca_18122 322 T 0 185
LOTGIsca_18122 323 A 0 186

Now I am very confused, as I thought pileup output files were either 5 or 10 columns, and I have 8... Also, I tried to filter the pileup output file with VARSCAN, using default parameters, and I get an output file with 0 lines.

Any input on what I have done, or what I should rather do instead would be greatly appreciated.

Thanks!
Erica
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Old 04-22-2016, 03:22 AM   #2
dpryan
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Which version of samtools are you using in Galaxy? If you look at the top of the page for it in Galaxy, you'll see something like "(Galaxy Version 2.1.1)".
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Old 04-22-2016, 04:57 AM   #3
erica.n
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Hi,

I am using Galaxy 16.04.rc1 -I'm not sure how to use another version..
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Old 04-22-2016, 05:05 AM   #4
dpryan
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The version of the tool, not the version of Galaxy itself (the two are independent). Like I said, click on the tool and see what tool version is printed at the top of the page.
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Old 04-22-2016, 05:12 AM   #5
GenoMax
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@erica.n: Are you using public galaxy server at Penn State or a local/other mirror elsewhere?
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Old 04-22-2016, 06:15 AM   #6
erica.n
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Hi,

Sorry I am using SAMtools v.2.1.1 and VarScan v.0.1

And I am on the galaxy server at Penn State.

thanks!
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Old 04-22-2016, 09:34 AM   #7
SNPsaurus
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By merging the bam files, you are going to collapse them all into one "sample" in the view of mpileup. Is that what you want? When we process RAD-type data we give it a list of bam files, one per sample.

The mpileup format is:
chromosome, 1-based coordinate, reference base, the number of reads covering the site, read bases and base qualities.
I see a chromosome, coordinate (322), ref base (T), # of reads (0)
LOTGIsca_18122 322 T 0 185

Is the option to report read position on? That might explain the 185, 186 in your output, although with 0 reads it would have to be a low quality base that is not shown but the position is.
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