Question: Read counts mapping against rrna genes in prokaryotic RNA-Seq experiment
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gravatar for buthercup_ch
12 months ago by
buthercup_ch20
Japan
buthercup_ch20 wrote:

Hello to everyone.

In my group, we are using RNA-Seq to study the expression of bacteria under different conditions, isolated or as dual-transcriptome when in association with plants.

When checking the table of total counts for each gene, I can observe an important number of reads mapping against the rrna genes, mostly 16S and 23S. I also found a similar amount of read counts mapping against rrnas when analyzing a dual experiment where no bacteria was present, just plant material.

Illumina reads from HiSeq200 were treated by TopHat and Cufflinks. Library preparations followed a RiboZero Gold treatment + TruSeq Stranded Total RNA kit. Additionally, rrna depletion was verified by running Bioanalyzer.

Have any one an idea where this mapping might come?

Thanks in advance for your help.

ADD COMMENTlink modified 12 months ago • written 12 months ago by buthercup_ch20
1

What fraction of the reads are aligning to rRNA? Experimental rRNA depletion may never be perfect so as long as the % is small it should be ok.

ADD REPLYlink modified 12 months ago • written 12 months ago by genomax33k
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gravatar for benformatics
12 months ago by
benformatics150
benformatics150 wrote:

Bacterial contamination is extremely common in many lab reagents.

https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-014-0087-z

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882852/

ADD COMMENTlink modified 12 months ago • written 12 months ago by benformatics150
0
gravatar for buthercup_ch
12 months ago by
buthercup_ch20
Japan
buthercup_ch20 wrote:

Hello

Thanks for your comments.

I was also thinking about if these sequences matching the bacterial rrna genes could actually be product of high sequence duplication (checking the FASTQC file for the samples showed red warning in this point). They might be an artifact product of PCR enrichment, over-sequencing, or contamination of library preparation (I just read about the highly frequent as common library prep and sequencing reagents gets contaminated by bacterial DNA).

What do you think?

Regards

Cristina

ADD COMMENTlink written 12 months ago by buthercup_ch20
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