Large-scale simultaneous measurement of epitopes and transcriptomes in single cells
2017 · paper · Biomedicine
Introduces CITE-seq, which uses oligonucleotide-labeled antibodies to measure cell-surface proteins and transcriptomes together in single cells through sequencing. The method works with existing single-cell sequencing approaches and gives more detailed cell characterization than RNA sequencing alone.
3 findings
Substance The 10× methods say that for CBMC profiling "~7,000 cells were loaded to obtain a yield of ~4,000 cells", but the analysis reports "human: 8005, mouse: 579, mixed: 33" barcodes after filtering, more than the number loaded. The Results also call the 8,005 human cells "8,005 cells in total".
- p. 8 Methods, 10× – CITE-seq
~7,000 cells were loaded to obtain a yield of ~4,000 cells
- p. 10 Methods, CBMC RNA normalization
human: 8005, mouse: 579, mixed: 33
- p. 4 Results
ADT profiles from 8,005 cells in
- p. 4 Results
from 8,005 cells in total
Only the authors can say what is right here.
Judge's note: I checked p.8, p.10 and p.4 on the page images. The p.11 neighbourhood of 40 cells is described as 0.5% of all cells, which fits about 8,000 cells, not 4,000. The paper mentions no multiple lanes or runs, so the stated load and yield do not match the reported data, and only the authors can reconcile them. The 'in total' wording also leaves out the 612 mouse and mixed barcodes.
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Reference or label Affiliation 3 ("New York University Center for Genomics and Systems Biology, New York, NY") is word-for-word the same as affiliation 2, yet P.K. Chattopadhyay is given a separate superscript 3.
- p. 1 Affiliations
3New York University Center for Genomics and Systems Biology, New York, NY
Only the authors can say what is right here.
Judge's note: Checked on the page 1 image: lines 2 and 3 of the affiliations are identical. Giving one institution two numbers is an error either way. Only the authors can say what Chattopadhyay's real affiliation is. The acknowledgements thank the Vaccine Research Center for instrument time, but that does not establish his affiliation.
Second check: another model agreed
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Reference or label The Data availability statement gives a placeholder instead of an accession number: "deposited to the gene expression omnibus (GEO) with the accession code XXXXXX."
- p. 1 Data availability
with the accession code XXXXXX.
Only the authors can say what is right here.
Judge's note: The placeholder is visible on the page 1 image. Readers cannot locate the deposited data, and only the authors can supply the accession number.
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13 minor (typos and the like; most checked by the judge only)
Reference or label "the centered logratio (CLR) transformation(Aitchison 1989)" is an author–year citation in a paper that otherwise uses numbered references, and Aitchison does not appear in the reference list.
- p. 11 Methods, CBMC ADT normalization
transformation(Aitchison 1989)
Judge's note: The p.11 image shows the author–year citation. A search of the full text finds 'Aitchison' only at this one place, so there is no matching entry in the numbered reference list.
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Wording The amount of 3T3 cells spiked into the CBMCs is given as "~4% 3T3" (Results, p.4) and "(~4%)" (10× methods, p.8), but as "~5% 3T3 cells were mixed into CBMC pool" (Cell culture, p.9). The doublet model then uses "a fixed mouse fraction of 6.5%".
- p. 4 Results
spiked a low number of mouse fibroblasts (~4%
- p. 8 Methods, 10× – CITE-seq
mouse cells at low frequency (~4%)
- p. 9 Methods, Cell culture
ins ~5% 3T3 cells were mixed into CBMC pool
- p. 4 Results, CBMC paragraph
mouse fibroblasts (~4%
Only the authors can say what is right here.
Judge's note: I read all three passages on the page images. The ~4% and ~5% values both describe the nominal input, and the paper does not reconcile them. The 6.5% figure is a model parameter and roughly matches the observed share of mouse barcodes (579 of 8617), so I do not count it as a defect. Both nominal values are approximate, so the discrepancy is minor, but only the authors can say which one is right.
Second check: another model agreed
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Wording The Results say "Murine cells clustered separately" right after describing the RNA-based clustering. The Fig. 3 legend says "The mouse control cell population was excluded from the clustering", and the methods say analysis was on human cells only.
- p. 4 Results
Murine cells clustered separately
- p. 16 Figure 3 legend
The mouse control cell population was excluded from the clustering.
Only the authors can say what is right here.
Judge's note: Checked on the page 4 and page 16 images and in the methods on p.10. The methods assign species from the fraction of UMIs, so the mouse cells were set apart by species assignment rather than by the clustering. The Results wording misdescribes this.
Second check: another model agreed
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Wording The CLR equation on p.11 ends at "ln(x5/g(x))" while the text says "there are only 13 components in this experiment". The last index should be generic (n) or 13.
- p. 11 Methods, CBMC ADT normalization, CLR equation
there are only 13 components in this experiment
Judge's note: The p.11 image shows the vector ending in x5/g(x). The sentence just before it states 13 components. This is a notational inconsistency, and the definition of CLR is otherwise clear.
Second check: another model agreed
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Typo Subject–verb agreement in the abstract: "as the throughput of these methods increase" should read "increases".
- p. 1 Abstract
as the throughput of these methods increase.
Judge's note: Seen on the page 1 image. The subject is 'throughput', which is singular.
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Typo A word is missing: "contain a barcode sequence for identification the antibody" should read "identification of the antibody".
- p. 2 Results, oligo design
barcode sequence for identification the antibody
- p. 2 Results, para 3
contain a barcode sequence for identification the antibody
Judge's note: Seen on the page 2 image.
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Typo Misspelling "Suppementary Fig. 3d,e"; it should be "Supplementary". On the same page, "Supplemental Fig. 3a,b" also differs from the paper's usual "Supplementary".
- p. 3 Results, CD8a paragraph
Suppementary Fig. 3d,e
Judge's note: Both are visible on the page 3 image, so this is not an extraction artefact.
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Typo Typographic inconsistencies: "CD8a intermediate(+) and CD8a low(+/−)" lacks a space before the parenthesis, and "(1ug per test" on p.9 uses 'ug' where p.8 has "1 µg per test".
- p. 3 CD8a pools
CD8a intermediate(+) and CD8a low(+/−)
- p. 9 Comparing flow cytometry
1ug per
Judge's note: Seen on the page 3 and page 9 images. The page 8 text has '1 µg per test' and '1 µg/µl', so the units are written inconsistently. This is cosmetic only.
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Typo Misspelled abbreviations: "For PMBC experiments cells were loaded at 150 cells/µL" (p.8; should be PBMC or CBMC) and "CMBC profiling experiment" (p.11; should be CBMC).
- p. 8 Methods, Drop-seq – CITE-seq
For PMBC experiments cells were loaded
- p. 8 Methods, Drop-seq – CITE-seq
For PMBC experiments cells were loaded at 150 cells/µL
- p. 8 Drop-seq methods
For PMBC experiments
- p. 11 Doublet estimation heading
CMBC profiling experiment
Judge's note: Both are seen on the page images. The 'Suppementary' misspelling also listed here is already covered by C005.
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Typo Misspellings: "CMBC profiling experiment" (p.11; CBMC everywhere else) and "Suppementary Fig. 3d,e" (p.3).
- p. 11 Methods, Estimation of doublet rate
CMBC profiling experiment
- p. 3 Results
Suppementary Fig. 3d,e
- p. 11
in our CMBC profiling experiment
Judge's note: I confirmed both misspellings on the page images: the p.11 Methods doublet section and the p.3 Results crop.
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Typo "we have shown that the CITE-Seq is fully compatible" has a stray article and capitalises the name as 'CITE-Seq'; elsewhere the paper writes 'CITE-seq'.
- p. 5 Discussion, final paragraph
we have shown that the CITE-Seq is fully compatible
- p. 5 Discussion
the CITE-Seq is fully compatible
Judge's note: Seen on the page 5 image. The same paragraph uses 'CITE-seq'.
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Typo Words are missing: "See Supplementary Table 2 for list antibodies" should read "for a list of antibodies".
- p. 6 Methods, List of Antibodies
See Supplementary Table 2 for list antibodies
Judge's note: Seen on the page 6 image.
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Typo A stray word: "cells were incubated in with mixtures of barcoded antibodies" should read "incubated with".
- p. 8 Methods, Cell staining
incubated in with mixtures of barcoded antibodies
Judge's note: Seen on the page 8 image.
Second check: none (judge only)