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ctlab / quant3p

A set of scripts for 3' RNA-seq quantification
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Peak calling error in SAMTools #2

Closed chandni177 closed 8 years ago

chandni177 commented 9 years ago

Hi,

I've been working on using quant3p for RNA-Seq data aligned using STAR. (Command below):

STAR --runThreadN 16 --genomeDir ./Genome --readFilesIn Barcode1.fastq --outFileNamePrefix MapBarcode1. --quantMode TranscriptomeSAM --outFilterMismatchNmax 6 --outFilterMultimapNmax 1

quant3p -n test -g gencode.vM6.annotation.gtf MapBarcode1.Aligned.toTranscriptome.out.bam

However, I always get the error SAMTools invalid BAM binary header (this is not a BAM file)

To investigate further, I tried running just the macs2-stranded on the same data and I still cannot get it to run. I figured it might be a problem with the BAM file from STAR (MapBarcode1.Aligned.toTranscriptome.out.bam) and I re-added the Header information into the BAM file. The error persisted.

Have you, by any chance, come across this? It would be great if you can help me out.

Thanks a lot, Chandni

assaron commented 9 years ago

Hi, Chandni,

quant3p (as does htseq) uses genomic alignment, not transcriptomic. So simple Aligned.out.bam should be used, not the Aligned.toTranscriptome one.

Also, here is the command we are using for STAR: "--outFilterMultimapNmax 15 --outFilterMismatchNmax 6 --outSAMstrandField All --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM" may be outSAMstrandField and outSAMtype options can make a difference.

chandni177 commented 9 years ago

I see, That helps. I will try it out.

Thanks a lot! Chandni

chandni177 commented 9 years ago

I tried the command that you suggested and used the Aligned.out.bam as input to quant3p :

quant3p/bin/macs2-stranded -n testQuant3p MapBarcode1.Aligned.out.sorted.bam Attached is the output (log). I still get the same error.

Thanks once again for helping out, Chandni

On Mon, Sep 21, 2015 at 4:07 PM, Alexey Sergushichev < notifications@github.com> wrote:

Hi, Chandni,

quant3p (as does htseq) uses genomic alignment, not transcriptomic. So simple Aligned.out.bam should be used, not the Aligned.toTranscriptome one.

Also, here is the command we are using for STAR: "--outFilterMultimapNmax 15 --outFilterMismatchNmax 6 --outSAMstrandField All --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM" may be outSAMstrandField and outSAMtype options can make a difference.

— Reply to this email directly or view it on GitHub https://github.com/ctlab/quant3p/issues/2#issuecomment-142108401.

assaron commented 9 years ago

Chandni, you didn't attach the log file.

Also, it would be great if you shared the resulting bam-file. For example, you can run samtools view -h MapBarcode1.Aligned.out.sorted.bam | head -n 10000 | samtools view -bS - > MapBarcode1.Aligned.out.sorted.h10K.bam and send me MapBarcode1.Aligned.out.sorted.h10K.bam.

chandni177 commented 9 years ago

I attached it on Gmail but I guess Github didn't take it.

Can I send it to you to a particular email address?

Thanks! Chandni

On Tue, Sep 22, 2015 at 1:33 PM, Alexey Sergushichev < notifications@github.com> wrote:

Chandni, you didn't attach the log file.

Also, it would be great if you shared the resulting bam-file. For example, you can run samtools view -h MapBarcode1.Aligned.out.sorted.bam | head -n 10000 | samtools view -bS - > MapBarcode1.Aligned.out.sorted.h10K.bam and send me MapBarcode1.Aligned.out.sorted.h10K.bam.

— Reply to this email directly or view it on GitHub https://github.com/ctlab/quant3p/issues/2#issuecomment-142375657.

assaron commented 9 years ago

The problem is in macs2-stranded expecting at least two bam files internally. Will fix soon.

chandni177 commented 9 years ago

Thanks for your help! Working perfectly fine with multiple files.