Question: Using bowtie2 to create BAM files to view strand-specific coverage in Artemis
0
gravatar for Chris S.
4.5 years ago by
Chris S.290
United States
Chris S.290 wrote:

I'm trying to map fastq files from bacterial RNA-seq studies and then view the bam files in Artemis using the coverage by strand or strand stack  view.  However, in the few paired-end examples I tried from the SRA, I just get reads mapping almost equally to both strands, so the coverage looks like a mirror image.  I also tried Rockhopper and the plus and minus tracks look right, but I would like to know how I can fix this code below.  Thanks.


#SEE http://www.ebi.ac.uk/ena/data/view/SRS430163
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR104/009/SRR1041589/SRR1041589_1.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR104/009/SRR1041589/SRR1041589_2.fastq.gz
gunzip *gz
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Yersinia_pestis_CO92_uid57621/NC_003143.gbk
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Yersinia_pestis_CO92_uid57621/NC_003143.fna

bowtie2-build NC_003143.fna yp
bowtie2 -p 4 -x yp -1 SRR1041589_1.fastq -2 SRR1041589_2.fastq | samtools view -bS - | samtools sort - tmh

samtools index tmh.bam
art -Dbam=tmh.bam NC_003143.gbk
# select views-> coverage by strand

rna-seq • 2.4k views
ADD COMMENTlink modified 3.7 years ago by gufernandez1010 • written 4.5 years ago by Chris S.290

Are you sure that's a strand-specific dataset? If it isn't, then what you're describing would be expected.

ADD REPLYlink written 4.5 years ago by Devon Ryan88k

Yes, these datasets are all small RNA studies that have identified antisense RNAs.  Also, Rockhopper seems to separate out the strands without problems using default settings, so I'm guessing there's some way to do that with bowtie.

ADD REPLYlink written 4.5 years ago by Chris S.290

Artemis has an option to filter reads and I just needed to select the first read in the pair.

ADD REPLYlink modified 4.4 years ago • written 4.5 years ago by Chris S.290
0
gravatar for gufernandez10
3.7 years ago by
Chile
gufernandez1010 wrote:

i knowis late, but i have the same trouble and i think that is a problem in artemis because i've been making some test getting the same problem every time, with different data and different alignment.

ADD COMMENTlink modified 3.7 years ago • written 3.7 years ago by gufernandez1010
0
gravatar for gufernandez10
3.7 years ago by
Chile
gufernandez1010 wrote:

sorry ,i'm a new user here, and i dont know exactly how post a new question or where.
 
 I'm using rockhopper to make my RNA-seq analysis with pair end data and a reference genome, i made the analysis, in the alignment step i got 78% mapped reads, all of them properly paired.  then I using Artemis as a genome browser that show all information about the paired and the mate ,i saw that the flag for each reads mapped sai that are mapped but with itself don't, for other data aligned with bwa a few reads 
mapped, but when i look the information with artemis i can see the same flag that represent a pared and the corresponding mate read. I really don't know why is happening, if it's a normal result or what?

Sorry again for my bad way of ask

ADD COMMENTlink modified 3.7 years ago • written 3.7 years ago by gufernandez1010
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