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Old 03-28-2013, 12:20 PM   #1
Pennaki
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Default Illumina Miseq bam file

Hi all!

I'm a newbie in NGS, trying to write a thesis on BRCA mutations on breast cancer, not a biologist, so please excuse my ignorance!

I got one .bam file, of a sample sequenced by Miseq of Illumina and even after indexing it using samtools I see no alignment in IGV. When I used tview in samtools, a string of N's appeared and no bases, nowhere.

Do I need something more than this bam file?

Thank u!
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Old 03-28-2013, 01:16 PM   #2
swbarnes2
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You need the reference it was aligned to, and that needs an index. samtools faidx can make that. In fact, remaking the reference faidx is a quick and easy troubleshooting step that you should always do if you think there might be a problem with your reference being properly recognized. It only takes a second to do, but a lot of software will take for granted that if a file of the right name exists, that it is the correct file, and not flawed. And when that's not the case, that software will behave poorly, without hinting at why.
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Old 03-28-2013, 01:51 PM   #3
BAMseek
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Try looking at the first few lines of the bam file

Quote:
samtools view input.bam | less -S
The 3rd and 4th columns are the chromosome and position. Pick one of those locations and go to it in IGV to see if you can find that read. Maybe run samtools flagstat to quickly see how many reads and alignments are in that BAM file.
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Old 03-29-2013, 09:53 AM   #4
Pennaki
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Thank u both for answering!

@BAMseek: I tried to read the file but all I got was blank with an "END". :/

@swbarnes2: I have only this bam file and 2 other fastg.gz files, which I was given after the sequencing from the instrument. Do I have to download a human genome reference?
I tried to view another example bam file and it worked perfectly on IGV and on samtools when I tview-ed it!

P.
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Old 03-29-2013, 12:00 PM   #5
BAMseek
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Quote:
@BAMseek: I tried to read the file but all I got was blank with an "END". :/
That's not good. What is the size of your BAM file? Also, try "samtools view -h <input.bam>" (the -h flag will also print out header information). Getting nothing might because the file is blank (or only has header information).
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Old 03-29-2013, 02:59 PM   #6
Pennaki
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Quote:
Originally Posted by BAMseek View Post
That's not good. What is the size of your BAM file? Also, try "samtools view -h <input.bam>" (the -h flag will also print out header information). Getting nothing might because the file is blank (or only has header information).
It's 237 MB. I tried samtools view -h <input.bam> and I get a zillion lines of bases, it's not empty!

Last edited by Pennaki; 03-29-2013 at 03:02 PM.
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Old 03-29-2013, 03:50 PM   #7
BAMseek
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Hmm, it's odd that samtools view gives you nothing but samtools view -h gives you lots of lines. So can you take one of those positions, and go to it in IGV? Also, what are the results of samtools flagstat <in.bam> ?
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Old 03-30-2013, 01:14 AM   #8
Pennaki
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Quote:
Originally Posted by BAMseek View Post
Hmm, it's odd that samtools view gives you nothing but samtools view -h gives you lots of lines. So can you take one of those positions, and go to it in IGV? Also, what are the results of samtools flagstat <in.bam> ?
I get this:

2033746 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
1997349 + 0 mapped (98.21%:nan%)
2033746 + 0 paired in sequencing
1016873 + 0 read1
1016873 + 0 read2
1894442 + 0 properly paired (93.15%:nan%)
1985672 + 0 with itself and mate mapped
11677 + 0 singletons (0.57%:nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

As I can see, it's chr13 and when I take it to IGV, i zoom and nothing!
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