Question: RPKM with bowtie output
0
gravatar for flogin
11 weeks ago by
flogin150
flogin150 wrote:

Hello guys,

There is a way to evaluate RPKM direct with bowtie 2 output (e.g. bam or sorted bam)? like an R package or some scripts to do this?

count bowtie rna rpkm • 200 views
ADD COMMENTlink modified 8 weeks ago by anoo0 • written 11 weeks ago by flogin150
2

Is this RNA-seq? Are you aware bowtie2 is not splice-aware? What organism? Do you have a GTF file or list of reference peaks (if not RNA-seq)?

ADD REPLYlink modified 11 weeks ago • written 11 weeks ago by ATpoint28k

It's a mapping of RNA-seq against a set of contigs, I just have the contigs, rna-seq files and bam files.

ADD REPLYlink written 11 weeks ago by flogin150
4
gravatar for swbarnes2
11 weeks ago by
swbarnes27.2k
United States
swbarnes27.2k wrote:

RSEM will take a bam file as input and return raw counts, RPKM and TPM

ADD COMMENTlink written 11 weeks ago by swbarnes27.2k

thanks swbarnes2, I'll check this.

ADD REPLYlink written 11 weeks ago by flogin150

Hello, Can you please provide the steps used for calculating expression using RSEM, after building the reference. The script is throwing errors.

ADD REPLYlink written 8 weeks ago by anoo0

Please read the manual and then come back with more specific questions while opening a new question, providing the necessary details.

ADD REPLYlink written 8 weeks ago by ATpoint28k

The script is throwing errors.

How do you expect us to help if you don't tell us what's going wrong?

ADD REPLYlink written 8 weeks ago by WouterDeCoster42k

I used the same commands given in the manual, with the BAM files I have,

software/RSEM-1.2.25/rsem-calculate-expression -p 8 --paired-end \ --bam \ --estimate-rspd \ --append-names \ --output-genome-bam \ exp/LPS_6h.bam \ ref/mouse_ref exp/LPS_6h

Error:

Warning: The SAM/BAM file declares less reference sequences (955) than RSEM knows (41078)! Please make sure that you aligned your reads against transcript sequences instead of genome. RSEM can not recognize reference sequence name 1!

"rsem-parse-alignments /home/user/data/RSEM/Rat /home/user/data/RSEM/Rat.temp/Rat /home/user/data/RSEM/Rat.stat/Rat /home/ec2-user/data/Rat/*.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

ADD REPLYlink written 8 weeks ago by anoo0
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