Question: Greetings!

I need some help with performing my rRNA decontamination step properly, which is part of pre-processing pipeline for my Illumina RNA-Seq reads, before mapping to the reference genome (a plant species).

**SOME RELEVANT LINKS AND MY CRUDE INFERENCES**:

https://www.biostars.org/p/65793/ - SILVA database is useful for human research, not so much for plants etc

https://www.biostars.org/p/266651/ - RFAM database searches will identify rRNA matches

https://www.biostars.org/p/325850/ - sormeRNA, and BBsplit are comparable tools useful for rRNA decontamination

https://www.biostars.org/p/184791/ - BBDuk can also be used

https://www.biostars.org/p/321714/ - BBMap is a tool I used, but output mapped (outm file) to rRNA fasta file, did not always correspond to rRNA sequences, not sure why

https://www.hindawi.com/journals/dpis/2013/854869/ - Eukaryotic SSU rRNA can have introns

**MY GOAL**:

Decontaminate RNA-Seq Illumina reads by removing rRNA sequences

**note:** I do have rRNA FASTA sequences from the genome annotation project (62 in all, and of different lengths) - but I am not sure whether they contain introns or not?

With these links, and my main inferences from those links listed above as background information, here are **MY QUESTIONS**:

1. Previous posts allude to how sormeRNA is much slower than BBSuite
tools, but that they should both give concordant results. Is there a
reason(s) to choose sormeRNA over BBsuite tools?

2. If not, within BBsuite tools, is there a good reason to choose one
over the other two ? (BBmap vs. BBsplit Vs BBduk) Should I check
for introns in the rRNA sequences, and if yes, then what's a good
method for that?

3. If any of the 62 rRNA sequences contain introns, then my
understanding is I remove the intronic sequences and used the
spliced rRNA sequences as the reference file for decontamination,
correct?

4. Are there common reason(s) why my BBmap trial returned outm mapped
to the rRNA reference FASTA file where some but not all contained
rRNA sequence match, as determined using online BLASTn at NCBI?

**note**: I can post syntax and more detail if this thread goes in the direction of BBmap, but thought that would distract the reader, so they are not included here yet..

I look forward to answers that cover all my questions 1 - 4. Thanks, in advance, for guidance from forum members.

Answer: BBDuk is a filter. At JGI we use it to remove reads with 31-mers corresponding to common ribosomal 31-mers, because some downstream tools like Trinity fail when there is an overload of rRNA reads.

Running BBDuk with common ribosomal kmers is good practice, but not ideal; the sensitivity and specificity are high, but not 100%. We do that at JGI because we work with metatranscriptomes for which the ribosomal sequences are not known and often 90% of the reads are ribosomal.

If you are doing RNA-seq on a reference organism, with a complete ribosomal sequence, the best practice IMO is to use BBSplit or Seal to split the reads between genomic and ribosomal, and then just run your analysis on the genomic reads. If you do not have a complete ribosomal sequence, you can still separate them with BBDuk. Mapping to Silva unfortunately takes too long.

I'm not a biologist. From my limited understanding of biology, it seems as though rRNAs are always unspliced. My assumption is that if you see introns in proks or mitos, they are simply misannotated. But don't consider me an authority here; I'm just relating what I see.