Is a transcriptome mapping rate of 55% acceptable for my total RNA experiments? How to check mapping rate of genome alignment?

[William Wright]

Hi everyone,

I'm using Bowtie2 with RSEM. Illumina RNA-seq (total, not mRNA-enriched) paired-end . My transcriptome mapping rates are around 55% for all samples. I also need genome mapping, and the "--output-genome-bam" is very handy. However, I can't see the mapping rate for the genome. 

Additionally - I'm a bit worried because I used STAR on these samples in the past to map to the genome (Hg19 then) with >90% mapping. 

I'm not sure if this is acceptable or normal or if I should continue or stop the analysis. 

Furthermore - I tried Salmon and the transcriptome mapping was also around 55% - so I don't think it's RSEM specific. Here's my code :

$rsem -p 8 --bowtie2 --bowtie2-path $bowtie2 --bowtie2-sensitivity-level sensitive --paired-end --strandedness reverse --append-names --sort-bam-by-coordinate --output-genome-bam $indir/CLEAN_Sample1_R1.fastq.gz $indir/CLEAN_Sample1_R2.fastq.gz $reference $outdir/RSEM_hg38__Sample1.fastq.gz

Thanks!

Charlie

[Peng]

Hi Charlie,

It seems to me that ~90% of your RNA-seq data mapped to genome (according to STAR) and ~55% of your RNA-seq data mapped to transcriptome (according to RSEM).  So there are ~35% of your RNA-seq data mapped to genomic regions without any transcript annotation.  Would this 35% something you expected?  Is it possible that this 35% could be largely ribosome RNA since your RNA-seq date is not mRNA-enriched? 

Best,

Peng

[William Wright]

Hi Peng,

thanks for the response. Indeed, it is possible that there is rRNA contamination, but I'm just not well-versed enough in transcriptome-mapping to know if this is what others get. How can I check the mapping rate of Bowtie2/RSEM's genome mapping step? 

Thanks!

Charlie

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[Peng]

Hi Charlie,

I don't know how to check the genome mapping rate for Bowtie2/RSEM.  If you use STAR/RSEM, you can get the genome mapping rate from STAR's 'Log.final.out' file.  Hope this information is helpful.

Best,

Peng

On Thursday, July 11, 2019 at 12:51:34 PM UTC-5, William Wright wrote:

Hi Peng,

thanks for the response. Indeed, it is possible that there is rRNA contamination, but I'm just not well-versed enough in transcriptome-mapping to know if this is what others get. How can I check the mapping rate of Bowtie2/RSEM's genome mapping step? 

Thanks!

Charlie

On Thu, Jul 11, 2019 at 12:10 PM 'Peng' via RSEM Users <rsem-...@googlegroups.com> wrote:

Hi Charlie,

It seems to me that ~90% of your RNA-seq data mapped to genome (according to STAR) and ~55% of your RNA-seq data mapped to transcriptome (according to RSEM).  So there are ~35% of your RNA-seq data mapped to genomic regions without any transcript annotation.  Would this 35% something you expected?  Is it possible that this 35% could be largely ribosome RNA since your RNA-seq date is not mRNA-enriched? 

Best,

Peng


On Thursday, July 11, 2019 at 11:55:49 AM UTC-5, William Wright wrote:

Hi everyone,

I'm using Bowtie2 with RSEM. Illumina RNA-seq (total, not mRNA-enriched) paired-end . My transcriptome mapping rates are around 55% for all samples. I also need genome mapping, and the "--output-genome-bam" is very handy. However, I can't see the mapping rate for the genome. 

Additionally - I'm a bit worried because I used STAR on these samples in the past to map to the genome (Hg19 then) with >90% mapping. 

I'm not sure if this is acceptable or normal or if I should continue or stop the analysis. 

Furthermore - I tried Salmon and the transcriptome mapping was also around 55% - so I don't think it's RSEM specific. Here's my code :

$rsem -p 8 --bowtie2 --bowtie2-path $bowtie2 --bowtie2-sensitivity-level sensitive --paired-end --strandedness reverse --append-names --sort-bam-by-coordinate --output-genome-bam $indir/CLEAN_Sample1_R1.fastq.gz $indir/CLEAN_Sample1_R2.fastq.gz $reference $outdir/RSEM_hg38__Sample1.fastq.gz

Thanks!

Charlie

--
RSEM website: http://deweylab.biostat.wisc.edu/rsem/
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