[Bioc-devel] Plans for multi-feature SingleCellExperiment?

Steve Lianoglou m@|||ng||@t@honeypot @end|ng |rom gm@||@com
Wed Jan 23 19:54:36 CET 2019

Hi all,

Thank you all for the quick and helpful replies.

For now, I'll just go with the default implementation in DropletUtils.
Thank you, Aaron, for keeping DropletUtils up to date with the updates
in Cell Ranger.

As an aside: speaking specifically about the updated 10x kit to
support these new assays, it's not so obvious to me that the biases
should be shared across these features. Given that, in the v3
chemistry (1) the mRNA pool and the "feature barcoded" pool are not
competing for the same capture probes on the gel beads; and (2) I
think the feature-barcoded libraries are PCR'd separately from the
mRNA pool as they come out "the other side" of the 10x toaster, it
doesn't seem strange to me that biases (and normalization factors)
might be different between the two feature spaces, but maybe I'm over
(or likely under :-) thinking things.

Thanks again!

Steve Lianoglou
Denali Therapeutics

On Tue, Jan 22, 2019 at 2:54 PM Aaron Lun <aaron.tin.long.lun using gmail.com> wrote:
> For 10X experiments, the Bioc-devel version of DropletUtils will read in
> the additional features as extra rows in the count matrix. This reflects
> how they are stored in the 10X output format. The row metadata will
> record the nature of the feature.
> In some cases it may be desirable to keep all the features together. For
> starters, it seems like many of the biases are likely to be shared
> (w.r.t. library preparation and capture efficiency), so one could
> imagine using the same scaling factors for normalization of both
> antibody-based features and endogenous mRNAs. In addition, all of the
> scater visualization methods rely on SCE inputs, so if you want to
> overlay them with protein marker intensities, they'll need to be in the
> same matrix.
> If you really need to only use mRNAs or antibody-based features, (i) you
> can explicitly subset the SCE based on the rowData, or (ii) pass a
> subsetting vector to the various scran/scater/whatever functions to tell
> them to only use the specified features. Admittedly, if you're going to
> be doing this a lot, it would be more convenient to form a MAE
> containing two SCEs so that you only have to pass the SCE you want into
> those functions.
> To that end I would be willing to entertain a PR to DropletUtils to
> create a MAE from an SCE. I'm more reluctant to add an isSpike()-like
> function. The rationale behind isSpike() was that spike-ins are constant
> across cells (theoretically) and thus a function could use this
> information to improve its calculations. It's less clear what
> mathematically useful information can be gained from protein markers -
> biological info, yes, but nothing that you would use to change your
> algorithm.
> -A
> Steve Lianoglou wrote:
> > Comrades,
> >
> > Sorry if I'm out of the loop and have missed anything obvious.
> >
> > I was curious what the plans are in the single-cell bioconductor-verse
> > to support single cell experiments that produce counts from different
> > feature-spaces, such as those produced by CITE-seq / REAP-seq, for
> > instance.
> >
> > In these types of experiments, I'm pretty sure we want the counts
> > generated from those "features" (oligo-conjugated Antibodies, for
> > instance) to be kept in a separate space than the mRNA counts. I think
> > we would most  naturally want to put these in something like an
> > `assay()` matrix with a different (rowwise) dimmension than the gene
> > count matrix, but that can't work since all matrices in the assay()
> > list need to be of the same dimensions.
> >
> > Another option might be to just add them as rows to the assay
> > matrices, but keep some type of feature space meta-information akin to
> > what `isSpike()` currently does;
> >
> > or add a new slot to SingleCellExperiment to hold counts from
> > different feature spaces, perhaps?;
> >
> > Or rely on something like a MultiAssayExperiment?
> >
> > Or?
> >
> > Curious to learn which way you folks are leaning ...
> >
> > Thanks!
> > -steve
> >
> > ps - sorry if this email came through twice, it was somehow magically
> > sent from an email address I don't have access to anymore.
> >

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