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Bacterial RNA-seq GC%

Hi,

I'm currently performing RNA-seq on bacterial samples (pseudomonas aeruginosa) and I'm getting the below per sequence GC content curve for around 10 of 30 samples. Essentially the (not shown here) other samples all gravitate around 65%, but there is a bump that gets bigger and bigger for different samples around the 55% mark and I'm struggling to determine what it is. The samples:

  • Are of great quality (also pictured below)
  • Have been trimmed with fastp, have a minimal length of 75bp and no apparent adapter contamination (according to fastQC anyway)
  • Have a higher sequence duplication level above ~500
  • All samples, including those with questionable GC content curve are mapping to the pseudomonas aeruginosa reference (self-assembly - by the group I'm doing this work for) at >=97%.

Does anyone know what it could be?

Good samples GC% Good samples GC%

Bad samples GC% Bad samples GC%

Good samples sequence dup Good samples sequence dup

Bad samples sequence dup Bad samples sequence dup

Good samples fastp GC% Good samples fastp GC%

Bad samples fastp GC% Bad samples fastp GC%

Good samples quality Good samples quality

Bad samples quality Bad samples quality

Thanks,
Matt

gc-content rna-seq bacterial
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written by fluentin44 • fluentin44

1 answer

It's probably rRNAs. 23S rRNA and 16S rRNA in Pseudomonas aeruginosa have a GC content of about 53% (https://www.ncbi.nlm.nih.gov/nuccore/NR_076166.1 , https://www.ncbi.nlm.nih.gov/nuccore/NR_026078.1). Some of your samples have lower quality of rRNA depletion than others.

• 0 views • link
written by shelkmike shelkmike

Thats a very convincing answer, thanks very much!

• 0 views • link
written by fluentin44 • fluentin44

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