RNA sequencing assay question

[hectorguzor]

Hello,

Question about 'RNA sequencing assay' (OBI:0001177) and 'RNA-seq assay' (OBI:0001271). In OBI, 'RNA-seq assay' is a subclass of 'RNA sequencing assay'. Are there examples of assays that fall under 'RNA sequencing assay' but not under 'RNA-seq assay'? Or are these just the same assay term?

Thanks

[bpeters42]

I suspect that this was a erroneously included duplicate entry. And it is probably my fault.

[mgiglio99]

Hi Hector - excellent question. For some info on this please see https://github.com/obi-ontology/obi/issues/1179 We made the parentage shift, but I have failed to get back to reviewing each of the child terms to see which should move up to 'RNA sequencing' parent. I feel there are some just in a quick look but I haven't done the legwork yet. Is this something you are interested in doing?

[hectorguzor]

@mgiglio99 thanks for pointing me to #1179. I can take a look at the child terms under "RNA-seq assay" to determine what should stay under it, and what (if any) should be moved as a subclass of "RNA sequencing assay".

[hectorguzor]

I looked into the subclasses of "RNA-seq assay" and identified the following as assays that could be moved under "RNA sequencing assay".

direct RNA sequencing assay PRO-cap isoform sequencing single-cell RNA sequencing assay

If someone can look into these and confirm I can make the changes to OBI.

Also, it was discussed on August 2's OBI call that the label ( and possibly the definition) of "RNA-seq assay" should be modified. I can also make those changes if someone could provide me with suggestions.

Thanks

[mgiglio99]

Hi Hector - thanks for looking into these. I agree with you on 'direct RNA sequencing assay' and 'PRO-cap'. However 'single-cell RNA sequencing assay' should stay a child of 'RNA-seq assay' - it's really just RNA-seq done from material from a single cell - there are amplification steps, but the general concept and workflow are the same. I'm not as sure about 'isoform sequencing' , but I think it likely should stay under 'RNA-seq assay' as well. It sounds like a form of RNA-seq optimized to provide long reads that cover the whole length of a transcript.

I'll try to come up with some suggestions for definition revisions.

[hectorguzor]

Discussed in OBI call 2021-08-16: 'direct RNA sequencing assay' and 'PRO-cap' should be moved as children of 'RNA sequencing assay'. The label for 'RNA-seq assay' should also be changed to reflect that it uses high throughput sequencing. Suggestion for new 'RNA-seq assay' was 'RNA high throughput sequencing assay', but still needs discussion.

[hectorguzor]

Discussed in OBI call 2021-09-21: Changes to the subclasses of 'RNA sequencing assay' as discussed in OBI call 2021-08-16 will be made in PR #1433. There will be no changes to the label of 'RNA-seq assay' as many people are familiar with that term. More discussion is needed if changes to the textual definition are to be made.

[mgiglio99]

For discussion on Nov. 1 call:

Proposal for new def for 'RNA-seq assay': Current def: "An RNA sequencing assay that determines an RNA sequence by analyzing the transcibed regions of the genome and or to quantitate transcript abundance." Proposed new def: "An RNA sequencing assay that employs high-throughput methods to assess the transcriptome, or a subset of a transcriptome (e.g. all mRNAs), from a given sample."

Also - another potential addition of an alternate name is "high-throughput RNA sequencing transcriptome assay"

[mgiglio99]

Revision to def draft after discussion on Nov. 1: "An RNA sequencing assay that employs high-throughput methods to profile transcription from a given sample." Decision is to use this definition. Not going to add another alternative label.

[mgiglio99]

Chris Mungall emailed about the possibility of aligning these terms with NCIT: Quoting the email: "Is there any attempt to coordinate with NCIT on 2? There are a lot of relevant terms, e.g SMRT sequencing http://purl.obolibrary.org/obo/NCIT_C146819" Asiyah is responding to Chris's email asking for more information on what he had in mind in terms of alignment. Possibly relevant work described here: http://obofoundry.org/ontology/ncit.html

[linikujp]

Hi, after searching the NCIt OBO/OWL version, I think this term should align to NCI "Whole Transcriptome Sequencing" http://purl.obolibrary.org/obo/NCIT_C124261. Please let me know if this makes more sense per Chris' request.

[bpeters42]

I have gone back through the history of comments on this term and related changes, and I have not found any written down arguments why 'RNA-seq assay' is different from 'RNA sequencing assay'. The initial push-back on merging the terms might have been due to the novelty of RNA-Seq by pyrosequencing / sequencing-by-synthesis vs. traditional Sanger sequencing. But if that is the distinction we want to make, we should put that in the definition. I would prefer to have a single RNA-sequencing assay term, that has RNA-Seq as a synonym.

On Mon, Nov 1, 2021 at 10:04 AM Asiyah Yu Lin @.***> wrote:

Hi, after searching the NCIt OBO/OWL version, I think this term should align to NCI "Whole Transcriptome Sequencing" http://purl.obolibrary.org/obo/NCIT_C124261. Please let me know if this makes more sense per Chris' request.

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[mgiglio99]

@linikujp I don't think the OBI 'RNA-seq' term is equivalent to NCIT 'whole transcriptome sequencing' because as we discussed today, RNA-seq is often used for studying just a portion of the transcriptome - for example just mRNA or just microRNA. The two terms are certainly related but NCIT has other related terms too including 'MicroRNA sequencing', 'mRNA sequencing', 'strand specific RNA sequencing' and 'non-coding RNA sequencing' - all of which fall under 'RNA-seq' in OBI. If NCIT were to group these terms under a parent then that parent would likely align to OBI's 'RNA-seq', but they don't.

@bpeters42 - the discussion on whether to merge 'RNA-seq' and 'RNA sequencing' was recorded in the https://github.com/obi-ontology/obi/issues/1179 issue. I agree, the notes are sparse. My memory is that we determined that there are RNA sequencing approaches that would not be considered RNA-seq because they aren't high-throughput and aren't focused on transcriptome profiling. We sorted those out a few weeks ago after Hector reviewed all of the RNA-seq children and some were moved up to be children of RNA sequencing. Do you feel we should revisit this decision?

[bpeters42]

Regarding merging RNA-seq and RNA-sequencing, I remember the same thing as you: that we had a good discussion and had a good reason to keep them separate. But I really can't remember what those reasons were. And we need to be able to document to outside users what makes us have a distinction. I don't see a clear distinction in the present proposed definitions and would propose to go back to the original merged one.

• Bjoern

On Mon, Nov 1, 2021 at 4:56 PM Michelle @.***> wrote:

@linikujp https://github.com/linikujp I don't think the OBI 'RNA-seq' term is equivalent to NCIT 'whole transcriptome sequencing' because as we discussed today, RNA-seq is often used for studying just a portion of the transcriptome - for example just mRNA or just microRNA. The two terms are certainly related but NCIT has other related terms too including 'MicroRNA sequencing', 'mRNA sequencing', 'strand specific RNA sequencing' and 'non-coding RNA sequencing' - all of which fall under 'RNA-seq' in OBI. If NCIT were to group these terms under a parent then that parent would likely align to OBI's 'RNA-seq', but they don't.

@bpeters42 https://github.com/bpeters42 - the discussion on whether to merge 'RNA-seq' and 'RNA sequencing' was recorded in the #1179 https://github.com/obi-ontology/obi/issues/1179 issue. I agree, the notes are sparse. My memory is that we determined that there are RNA sequencing approaches that would not be considered RNA-seq because they aren't high-throughput and aren't focused on transcriptome profiling. We sorted those out a few weeks ago after Hector reviewed all of the RNA-seq children and some were moved up to be children of RNA sequencing. Do you feel we should revisit this decision?

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[mgiglio99]

Hi @bpeters42 We can certainly revisit this. I was originally in favor of doing the merger when I first submitted https://github.com/obi-ontology/obi/issues/1179. But at the same time I could see the logic of doing the rearrangement that we eventually decided on. The two terms 'direct RNA sequencing assay' and 'PRO-cap', after review by Hector and me, were moved out from under 'RNA-seq assay' to be children of the (now) parent term 'RNA sequencing assay'. @hectorguzor should chime in here too - but I think we decided to move these because they did not follow the traditional RNA-seq process of RNA -> cDNA -> sequence. However, as I look at them again - I could make the argument that PRO-cap is a variety of RNA-seq. I think 'direct RNA sequencing assay' is more problematic - It's a very different process than regular RNA-seq. Not sure if it should be a child of RNA-seq. I guess it depends on how we end up defining RNA-seq. If we decide on a merger, which label would we use - 'RNA-seq' or 'RNA sequencing assay'? I guess this can go on the agenda for discussing again.

looping in @nsuvarnaiari

[bpeters42]

First of all I want to apologize for complaining here and not being on Monday's call. It just struck me that whatever definitions we come up with must be very clear on what the difference between the terms is, and I currently don't see that.

If we do merge the term, I would vote for having the label be 'RNA sequencing assay', and have 'RNA-Seq assay' be an alternative label.

On Mon, Nov 1, 2021 at 7:48 PM Michelle @.***> wrote:

Hi @bpeters42 https://github.com/bpeters42 We can certainly revisit this. I was originally in favor of doing the merger when I first submitted #1179 https://github.com/obi-ontology/obi/issues/1179. But at the same time I could see the logic of doing the rearrangement that we eventually decided on. The two terms 'direct RNA sequencing assay' and 'PRO-cap', after review by Hector and me, were moved out from under 'RNA-seq assay' to be children of the (now) parent term 'RNA sequencing assay'. @hectorguzor https://github.com/hectorguzor should chime in here too - but I think we decided to move these because they did not follow the traditional RNA-seq process of RNA -> cDNA -> sequence. However, as I look at them again - I could make the argument that PRO-cap is a variety of RNA-seq. I think 'direct RNA sequencing assay' is more problematic - It's a very different process than regular RNA-seq. Not sure if it should be a child of RNA-seq. I guess it depends on how we end up defining RNA-seq. If we decide on a merger, which label would we use - 'RNA-seq' or 'RNA sequencing assay'? I guess this can go on the agenda for discussing again.

looping in @nsuvarnaiari https://github.com/nsuvarnaiari

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[hectorguzor]

Discussed in OBI 2022-05-16: We need to dedicate the majority of a call to this issue. In preparation, prior to the call people should look at the history of this issue and also take a look at #1179 . We should also look at transcription profiling assay to determine how this branch should be connected to the 'RNA sequencing assay' branch.

[mgiglio99]

Notes from Oct. 17, 2022 call on this topic: (notes were posted in the agenda/notes doc right after the call, but there was a delay in posting them here - Michelle's fault.) -We discussed how the output of the assay should define the axes on which we organize. Output being a “profile” or sequence data. -from Chris in chat: Assay = Sample processing + sequencing technique + assay objective -We decided to assert the “profile” (as the assay objective) plus sample prep aspect of the assay and keep sequencing technique out and capture as ‘detection technique’. -Thus terms will be collected together if they are doing any kind of transcription profiling. We decided that examining the expression of even one gene counts as a profile. Thus terms under ‘transcription profiling assay’ will include both high-throughput and low-throughput methods. -Currently ‘transcription profiling assay’ has children for things other than sequencing such as microarray and RTPCR, thus sequencing would be one other way of transcription profiling and we could create a grouping term such as ‘transcription profiling by RNA sequencing’. -In the case of the example term ‘polyA-selected RNA sequencing assay’, which is currently a child of ‘RNA-seq assay’, this term is combining sample prep and sequencing. In the new structure this term would move to be a child of ‘transcription profiling assay by RNA sequencing’ and would be renamed to something like: ‘polyA-selected mRNA profiling assay’ and would have ‘RNA sequencing assay’ (or perhaps ‘RNA-seq assay’ - see below) as the detection method. -Suggested revision to ‘transcription profiling assay’ definition: “An assay that determines the presence or abundance of specific RNA molecules in an input sample” (Bjoern) -Suggested definition for RNA sequencing: “An assay that determines the RNA sequence of molecules in an input sample” (Bjoern) -One thing we didn’t solve is how to deal with ‘RNA-seq assay’ vs. ‘RNA sequencing assay’ - should they both be kept or just one and if just one, which one? There was discussion of adding RNA-seq to term names whenever possible to indicate that. There was discussion of alternate names with RNA-seq. There was mention of how a “narrow” or “related” synonym might be useful in this case (but OBI doesn’t have those.) Some suggestions focused on defining RNA-seq based on sequencing technology, but there was concern that multiple technologies actually fall under RNA-seq. RNA-seq is not a precise term and is used in many contexts. -We decided to punt on the RNA-seq vs. RNA sequencing question for now - but we’ll go ahead and try to sort out the children of ‘transcription profiling assay’ and ‘RNA-seq assay’ according to the design pattern above and we’ll revisit later.

[mgiglio99]

Discussed on call on Jan. 23 Michelle proposed a possible structure in this google doc: https://docs.google.com/document/d/1ybbTA_-izcr5X4aC-BgT0NCPIKwU5_7FI_Z_eoGG3w0/edit?usp=sharing Discussion Notes: The consensus was to try the structure proposed in the google doc in which one axis is focused on assay objective (e.g. transcription profiling) and the other on the technique that was used (e.g RNA sequencing). All of the existing children under transcription profiling and RNA sequencing/RNA-seq will need to be examined to see where they belong. This will likely get tricky, but will test whether this structure works. Another decision was that the term that would correspond to the existing RNA-seq term (and have an alternate label that is ‘RNA-seq’) would be ‘transcription profiling by RNA sequencing’ (which includes both cDNA and direct RNA sequencing) as it was felt that this term best encompasses the community use of the term RNA-seq. Some of the children currently under RNA-seq are not involved in transcription profiling and will be rehomed - for example ‘ribosomal profiling by sequencing assay'. This term is for RiboSeq that is actually a translation profiling approach. Thus the term will be renamed to 'translation profiling by RiboSeq' (or something like that) and moved to be a child of 'translation profiling assay', with 'RNA sequencing assay' as a detection technique. This example was added to the google doc. Another example added was one to go with ‘transcription profiling by RT-PCR assay’, another term under ‘transcription profiling’. This term would have ‘polymerase chain reaction assay’ as a detection technique. Michelle, Suvvi, Chris, Hector, and Dan will start to work on sorting out the child terms in these nodes.

[mgiglio99]

Discussed on May 13th, 2024 call. We are going to get this started back up again - hopefully starting near the end of the summer. Sebastian is going to take Hector's place to help on this effort and Dan is willing to continue. Michelle and Suvvi will also continue.