how to make a .bam file from fastQ for RNA-seq ion torrent with BBmap
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5.2 years ago
genya35 ▴ 40

Hello,

Could someone please help with the exact syntax to make bam file from fastQ RNA-seq ion torrent with BBmap installed on windows 7 (command prompt). I've installed BBmap on Windows 7 but struggling to understand how to run the tool.

I found a post that describes this line but it's not clear how to provide the reference for the alignment?

I've tried indexing the reference but it's not working:

1. I navigated to bbmap folder where bbmap.sh and hg19.fasta are located and ran the following command: C:\tesm\bbmap>bbmap.sh ref=hg19.fasta and 'Open With' window popped up. What am I doing wrong?

Thank you.

RNA-Seq • 2.4k views
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I haven't done this, and certainly not on Windows, but my guess would be you need the following

C:\tesm\bbmap\bbmap.sh ref=hg19.fasta

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Looks like shell doesn't run on Windows after all. Although, it said in the documentation that BBmap can be ran from Windows. I will try to install on Linux server next. Could you please advice how to execute these commands on Linux?

Thanks

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It seems to be working well on Linux. Could you please suggest which settings to use for RNA-Seq Ion Torrent reads? Thank you very much.

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For human RNA-seq you should add the flag "maxindel=400k". You don't need to change anything else.

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C: pindel not picking up on a lab-made gene knockout deletion

Unfortunately, the shellscripts don't work in Windows (at least, not my version of Windows, though I've heard that Windows 10 can support them...) so the syntax changes a little compared to Linux.

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Unfortunately, the forum is not allowing me to add another comment so I'm replacing the existing comment.

@Brian,

Thank you so much for your help.

I ran the given command and it produced a sam file. Next, I used samtools to make a bam and index the bam. The problem occurred during the indexing. It gives me an error "NO_COOR reads not in a single block at the end 3 -1". Is there anything you can suggest to tweak in your tool parameters so no such errors are produced? I'm working with Ion Torrent Oncomine RNA data.

Thank you for your help again.

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Yes, it can be run in Windows; the command is:

java -cp C:\temp\bbmap\current align2.BBMap -Xmx26g in=reads.fq ref=hg19.fasta out=mapped.sam maxindel=400k


Whereas in Linux the command would be

bbmap.sh -Xmx26g in=reads.fq ref=hg19.fasta out=mapped.sam maxindel=400k


If samtools is in your path you can name the output file "mapped.bam" and a bam file will be created instead of a sam file.

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I forgot to mention that I'm trying Bbmap out on Ion Torrent (Oncomine) human targeted fusion data. I'm not sure if parameters would have to be set differently in this case? I've converted unmapped bam to fastq, ran your tool to make a sam then used samptools to make a bam, sorted and indexed the file. Your tool ran very fast without problems and made a small bam file. However, it looks like none of the reads aligned since I see nothing in IGV when I look at the bam. I'm hoping to identify the breakpoints, both mapped and unmapped reads. Is there anything else you would suggest to get the alignment to work with your tool?

Thank you

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BBMap should not need any special parameters for Ion Torrent data. Perhaps you could post your command and the stderr output?

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The alignment worked. I had trouble seeing it in IGV because few reads aligned. I also ran the same fastq through hisat2 and your tool performed equally well. Thank you very much.

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I can't really help you if you don't answer my questions. It sounds likely that you have contamination. I suggest you try BLASTing some reads to see what they hit.

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@Brian I'm trying to use your tool by aligning RNA fastq reads to a small fasta file I've made that contains the junctions of two genes where I know the fusion has occurred. I've created a fasta with >header and pasted the DNA sequence. There was no output. Could you please suggest if I need to make modifications to the file to get it to work? Or this this not going to work at all? Thanks

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I got it to make sam file but samtools crashes when I tried to sort that file.

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samtools crashes when I tried to sort that file.

Always include the command you used and the full error message when you are reporting something that doesn't work as expected.