Spatially resolved transcriptomics (SRT) provide gene expression close to, or even superior to, single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, SRT expression data suffer from high noise levels, due to the shallow coverage in each sequencing unit and the extra experimental steps required to preserve the locations of sequencing. Fortunately, such noise can be removed by leveraging information from the physical locations of sequencing, and the tissue organization reflected in corresponding pathology images. In this work, we developed Sprod, based on latent graph learning of matched location and imaging data, to impute accurate SRT gene expression. We validated Sprod comprehensively and demonstrated its advantages over previous methods for removing drop-outs in single-cell RNA-sequencing data. We showed that, after imputation by Sprod, differential expression analyses, pathway enrichment and cell-to-cell interaction inferences are more accurate. Overall, we envision de-noising by Sprod to become a key first step towards empowering SRT technologies for biomedical discoveries.
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The Visium datasets are obtained from the public 10X resources/datasets website: https://www.10xgenomics.com/resources/datasets. The IDs of the datasets are: human-lymph-node-1-standard-1-1-0, Human-ovarian-cancer-whole-transcriptome-analysis-stains-dapi-anti-pan-ck-anti-cd-45-1-standard-1-2-0, human-ovarian-cancer-targeted-pan-cancer-panel-stains-dapi-anti-pan-ck-anti-cd-45-1-standard-1-2-0 and human-breast-cancer-block-a-section-1-1-standard-1-1-0. The ID of the standard 10X scRNA-seq dataset used in Fig. 1c is 10-k-peripheral-blood-mononuclear-cells-pbm-cs-from-a-healthy-donor-single-indexed-4.0.0. The Slide-Seq data are available from the publicly archived data by Stickels et al.1. Specifically, we used the Puck_200115_08 data from https://singlecell.broadinstitute.org/single_cell/study/SCP815/highly-sensitive-spatial-transcriptomics-at-near-cellular-resolution-with-slide-seqv2.
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We acknowledge the ENCODE Consortium and the ENCODE production laboratories that generated the eCLIP datasets used in our study. We acknowledge J. Johnson for providing input on the interpretation of the mouse Slide-Seq data. This study was supported by the National Institutes of Health (NIH) (5P30CA142543 to T.W., G.X. and Y.X., 1R01CA258584 to T.W., U01AI156189 to T.W. and Y.X., R01DE030656 to G.X., R01GM141519 to G.X., R01GM140012 to G.X., U01CA249245 to G.X., R35GM136375 to Y.X., 2P50CA070907 to T.W., Y.X. and G.X., R01AG075582 to L.W., 3U01AI156189-01S1 to T.W.), National Science Foundation (NSF DMS-2009689 to L.W.), and Cancer Prevention Research Institute of Texas (CPRIT RP190208 to T.W. and RP190107 to G.X.).
The authors declare no competing interests.
Peer review information
Nature Methods thanks Nikos Karaiskos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary handling editor: Lin Tang, in collaboration with the Nature Methods team.
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Extended Data Fig. 1 Correlation between PTPRC RNA expression (targeted) and CD45 protein expression (IF).
Red arrows mean that the results are limited to spots of higher quality. Yellow arrows mean that the single gene of PTPRC is replaced by a signature of PTPRC by including correlated genes.
Gene expression clustering of the beads in the mouse brain Slide-Seq dataset reflects the multi-cellular structures of mouse brain hippocampus. a, Slide-seq dataset puck 200306_03 and b puck 200115_08 by Stickels et al.1.
Extended Data Fig. 4 Deviances between CD45 IF intensities and the expression levels of PTPRC (left: original, right: denoised).
CD45 IF intensities and PTPRC expression values were normalized and distributionally warped to the same scale so they can be directly compared. The differences between CD45 IF and PTPRC on each spot are denoted by color. Red refers to small differences and green refers to larger differences.
Extended Data Fig. 5 Spatial IgD expression of the raw Visium data (left) and the Sprod-adjusted data (right).
The red circles mark the mantle zone to be highlighted in Fig. 3d.
Extended Data Fig. 6 Spearman correlations between IgD and CD3/CD20/CD1c for the human lymph node Visium dataset.
Results are shown for the original expression data, SAVER/scImpute-corrected data, the Sprod-corrected data, and the Sprod-corrected data with image/location information scrambled.
The four tumor regions (blue, green, orange and red) that were extracted, according to expressional clustering and concordance with the H&E stained slide.
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Wang, Y., Song, B., Wang, S. et al. Sprod for de-noising spatially resolved transcriptomics data based on position and image information. Nat Methods 19, 950–958 (2022). https://doi.org/10.1038/s41592-022-01560-w