DOI: 10.5061/dryad.37pvmcvsg
title: MERFISH and snRNAseq analysis of healthy and disease human liver

description: Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of
 cell types and their heterogeneity within the human liver, but the spatial
 organization at single-cell resolution has not yet been described. Here we
 apply multiplexed error robust fluorescent in situ hybridization (MERFISH)
 to map the zonal distribution of hepatocytes, spatially resolve subsets of
 macrophage and mesenchymal populations, and investigate the relationship
 between hepatocyte ploidy and gene expression within the healthy human
 liver. We next integrated spatial information from MERFISH with the more
 complete transcriptome produced by single-nucleus RNA sequencing
 (snRNA-seq), revealing zonally enriched receptor-ligand interactions.
 Finally, MERFISH and snRNA-seq analysis of fibrotic liver samples
 identified two hepatocyte populations that expanded with injury and do not
 have clear zonal distributions. Together these spatial maps of the healthy
 and fibrotic liver provide a deeper understanding of the cellular and
 spatial remodeling that drives disease which, in turn, could provide new
 avenues for intervention and further study.
description: Two measurement modalities were used to generate these data,
 including multiplexed error robust fluorescence in situ hybridization
 (MERFISH) and single-nucleus RNA sequencing (snRNAseq).
description: # MERFISH and snRNAseq analysis of healthy and disease human liver Brianna
 Watson, Biplab Paul, Jeffrey Moffitt, and Alan Mullen 2024 ## Anndata
 structures Multiple anndata structures are provide as h5ad files. These
 anndata structures were generated with the scanpy pipeline and can be
 loaded in python with the associated tools integrated into the pipeline.
 The contents of these anndata structures are also provided as csv files
 for convience. These include: (1) adata_healthy_merfish.h5ad (2)
 adata_healthy_diseased_merfish.h5ad (3)
 adata_merfish_nucseq_healthy_hep.h5ad (4) adata_healthy_nucseq.h5ad (5)
 adata_healthy_diseased_nucseq.h5ad (6)
 adata_merfish_nucseq_diseased_hep.h5ad Each anndata frame contains
 distinctive values for the respective data set. A description for each
 will be found below. ### Convenience csv files For convenience, we have
 also exported important elements of each anndata structure as a csv file.\
 These files share the same name as the anndata structures. They are stored
 in the anndata_fields folder. Files starting with
 'cell_properties_' contain all metadata associated with the
 fields defined below. Files starting with 'X_' contain the
 normalized count matrix. Files starting with 'X_raw' contain the
 raw count matrix. Files starting with 'gene_names' contain the
 names of genes in the order they are quantified within the count matrices.
 ### Anndata descriptions #### (1) adata\_healthy\_merfish.h5ad ``` This
 structure contains only data from healthy patient samples which were
 imaged with the MERFISH technique. Raw data is stored in the adata.raw.X
 while adata.X is normalized by the total counts per cell, scaled to a
 uniform value, and then converted to logarithmic space by adding a
 pseudocount and applying a natural log transform. The obs field contains
 the properties of each observed cell and for this structure are defined as
 follows (this is stored as 'cell_properties' in the csv format)
 -sample_id: identification of patient -condition: defines condition of
 patient, all samples in this set were healthy -cell: cell id, values
 restart for each experiment -density: number of RNA per area -area: area
 of cell in microns^2 -x: x-position of cell centroid in microns -y:
 y-position of cell centroid in microns -dapi_intensity: intensity of DAPI
 stain for nuclei which were found to have 55% of nuclei within
 cellpose-baysor boundary from central z-plane -nuclei: number of nuclei
 attributed to cell; nuclei boundary was required to have 55% within
 cellpose-baysor boundary -batch: product of experiment concatenation; is
 unique identifier of experiment -n_counts: sum of counts within cell
 -log_count: natural log of n_counts -n_genes: number of genes for the cell
 -Cell_Type: final cell type annotations (as they appear in submitted text)
 -Additional ontology terms to describe patients data, cell types, etc. as
 defined by CellxGene -organism_ontology_term_id: NCBITaxon:9606 for human,
 NCBITaxon:10090 for mouse -donor_id: free-text identifier that
 distinguishes the unique individual that data were derived from (same as
 sample_id) -development_stage_ontology_term_id: HsapDv if human, MmusDv if
 mouse, unknown if information unavailable -sex_ontology_term_id:
 PATO:0000384 for male, PATO:0000383 for female, or unknown if unavailable
 -self_reported_ethnicity_ontology_term_id: HANCESTRO multiple
 comma-separated terms may be used if more than one ethnicity is reported.
 If human and information unavailable, use unknown. Use na if non-human.
 -disease_ontology_term_id: MONDO or PATO:0000461 for 'normal'
 -tissue_type: tissue, organoid, or cell culture -tissue_ontology_term_id:
 UBERON -cell_type_ontology_term_id: CL -assay_ontology_term_id: EFO
 -suspension_type: cell, nucleus, or na, as corresponding to assay. Base
 designation on cellxgene table. ("na" for this data set) ```
 #### (2) adata\_healthy\_diseased\_merfish.h5ad ``` This structure
 contains data from both healthy and diseased patient samples which were
 imaged with the MERFISH technique. Raw data is stored in the adata.raw.X
 while adata.X is normalized by the total counts per cell, scaled to a
 uniform value, and then converted to logarithmic space by adding a
 pseudocount and applying a natural log transform. The obs field contains
 the properties of each observed cell and for this structure are defined as
 follows (this is stored as 'cell_properties' in the csv format)
 -sample_id: identification of patient -condition: defines condition of
 patient, either healthy or diseased -cell: cell id, values restart for
 each experiment -density: number of RNA per area -area: area of cell in
 microns^2 -x: x-position of cell centroid in microns -y: y-position of
 cell centroid in microns -batch: product of experiment concatenation; is
 unique identifier of experiment -n_counts: sum of counts within cell
 -log_count: natural log of n_counts -n_genes: number of genes for the cell
 -Cell_Type_final: final cell type annotations (as they appear in submitted
 text) -Additional ontology terms to describe patients data, cell types,
 etc. as defined by CellxGene -organism_ontology_term_id: NCBITaxon:9606
 for human, NCBITaxon:10090 for mouse -donor_id: free-text identifier that
 distinguishes the unique individual that data were derived from (same as
 sample_id) -development_stage_ontology_term_id: HsapDv if human, MmusDv if
 mouse, unknown if information unavailable -sex_ontology_term_id:
 PATO:0000384 for male, PATO:0000383 for female, or unknown if unavailable
 -self_reported_ethnicity_ontology_term_id: HANCESTRO multiple
 comma-separated terms may be used if more than one ethnicity is reported.
 If human and information unavailable, use unknown. Use na if non-human.
 -disease_ontology_term_id: MONDO or PATO:0000461 for 'normal'
 -tissue_type: tissue, organoid, or cell culture -tissue_ontology_term_id:
 UBERON -cell_type_ontology_term_id: CL -assay_ontology_term_id: EFO
 -suspension_type: cell, nucleus, or na, as corresponding to assay. Base
 designation on cellxgene table. ("na" for this data set) ```
 #### (3) adata\_merfish\_nucseq\_healthy\_hep.h5ad ``` This structure
 contains data from only healthy patient samples which were imaged with the
 MERFISH or processed with snRNA-Seq. adata.X is normalized by the total
 counts per cell, scaled to a uniform value, then converted to logarithmic
 space by adding a pseudocount and applying a natural log transform, and
 finally z-scored. This processing was done for the MERFISH and snRNA-Seq
 seperatly before concatenating the two data sets together. The raw data
 was not included in the structure since the units would not be equivalent
 between the two techniques. The obs field contains the properties of each
 observed cell and for this structure are defined as follows (this is
 stored as 'cell_properties' in the csv format) -CellType:
 snRNAseq annotation of cell from snRNAseq analysis alone -n_genes: number
 of genes for the cell -sample_id: identification of patient (MERFISH
 measurements) -batch: product of anndata concatenation; is unique
 identifier of technique -cell_name: Hepatocyte annotation from analysis of
 MERFISH data alone -leiden_r1.5_joint: leiden resolution used for joint
 annotations -Hep_Type_Joint: final joint annotation of heaptocytes
 -sample_id_sn: identification of patient (snRNAseq measurements) ``` ####
 (4) adata\_healthy\_nucseq.h5ad ``` This structure contains data from only
 healthy patient samples which were processed with snRNA-Seq. Raw data is
 stored in the adata.raw.X while adata.X is normalized by the total counts
 per cell, scaled to a uniform value, and then converted to logarithmic
 space by adding a pseudocount and applying a natural log transform. The
 obs field contains the properties of each observed cell and for this
 structure are defined as follows (this is stored as
 'cell_properties' in the csv format) -Sex: biologically sex of
 patient (F or M) -Age: Age range of patient (ie. 51-60 years of age)
 -log10GenesPerUMI: number of genes detected per UMI -seurat_clusters:
 clustering of cell based on seurat parameters -CellType: cell annotations
 as determined by ScType -Condition: classification of tissue as healthy
 (normal) or fibrotic (disease) -n_counts: number of counts after mt
 filtering -log_counts: natural log of n_counts -n_genes: number of genes
 for the cell -cell_type_final: cell type annotations as the appear in
 final text for snRNA-Seq -sample_id: identification of patient -Additional
 ontology terms to describe patients data, cell types, etc. as defined by
 CellxGene -organism_ontology_term_id: NCBITaxon:9606 for human,
 NCBITaxon:10090 for mouse -donor_id: free-text identifier that
 distinguishes the unique individual that data were derived from (same as
 sample_id) -development_stage_ontology_term_id: HsapDv if human, MmusDv if
 mouse, unknown if information unavailable -sex_ontology_term_id:
 PATO:0000384 for male, PATO:0000383 for female, or unknown if unavailable
 -self_reported_ethnicity_ontology_term_id: HANCESTRO multiple
 comma-separated terms may be used if more than one ethnicity is reported.
 If human and information unavailable, use unknown. Use na if non-human.
 -disease_ontology_term_id: MONDO or PATO:0000461 for 'normal'
 -tissue_type: tissue, organoid, or cell culture -tissue_ontology_term_id:
 UBERON -cell_type_ontology_term_id: CL -assay_ontology_term_id: EFO
 -suspension_type: cell, nucleus, or na, as corresponding to assay. Base
 designation on cellxgene table. ("na" for this data set) ```
 #### (5) adata\_healthy\_diseased\_nucseq.h5ad ``` This structure contains
 data from healthy and diseased patient samples which were processed with
 snRNA-Seq. Raw data is stored in the adata.raw.X while adata.X is
 normalized by the total counts per cell, scaled to a uniform value, and
 then converted to logarithmic space by adding a pseudocount and applying a
 natural log transform. The obs field contains the properties of each
 observed cell and for this structure are defined as follows (this is
 stored as 'cell_properties' in the csv format) -Condition:
 classification of tissue as healthy (normal) or fibrotic (disease) -Sex:
 biologically sex of patient (F or M) -Age: Age range of patient (ex. 51-60
 years of age) -log10GenesPerUMI_injured: number of genes detected per UMI
 (from seurat analysis for injured cells) -seurat_clusters_injured:
 clustering of cell based on seurat parameters (from seurat analysis for
 injured cells) -CellType_injured: cell annotations as determined by ScType
 (from seurat analysis for injured cells) -cell_type_final_injured: cell
 type annotations as the appear in final text for injured snRNA-Seq
 -log10GenesPerUMI_healthy: number of genes detected per UMI (from seurat
 analysis for healthy cells) -seurat_clusters_healthy: clustering of cell
 based on seurat parameters (from seurat analysis for healthy cells)
 -CellType_healthy: cell annotations as determined by ScType (from seurat
 analysis for healthy cells) -cell_type_final_healthy: cell type
 annotations as the appear in final text for healthy snRNA-Seq -batch:
 product of anndata concatenation; is unique identifier of technique
 -n_counts: number of counts after mt filtering -log_counts: natural log of
 n_counts -n_genes: number of genes for the cell -sample_id: identification
 of patient -Additional ontology terms to describe patients data, cell
 types, etc. as defined by CellxGene -organism_ontology_term_id:
 NCBITaxon:9606 for human, NCBITaxon:10090 for mouse -donor_id: free-text
 identifier that distinguishes the unique individual that data were derived
 from (same as sample_id) -development_stage_ontology_term_id: HsapDv if
 human, MmusDv if mouse, unknown if information unavailable
 -sex_ontology_term_id: PATO:0000384 for male, PATO:0000383 for female, or
 unknown if unavailable -self_reported_ethnicity_ontology_term_id:
 HANCESTRO multiple comma-separated terms may be used if more than one
 ethnicity is reported. If human and information unavailable, use unknown.
 Use na if non-human. -disease_ontology_term_id: MONDO or PATO:0000461 for
 'normal' -tissue_type: tissue, organoid, or cell culture
 -tissue_ontology_term_id: UBERON -cell_type_ontology_term_id: CL
 -assay_ontology_term_id: EFO -suspension_type: cell, nucleus, or na, as
 corresponding to assay. Base designation on cellxgene table.
 ("na" for this data set) ``` #### (6)
 adata\_merfish\_nucseq\_diseased\_hep.h5ad ``` This structure contains
 data from healthy and diseased patient samples which were imaged with the
 MERFISH and diseased samples processed with snRNA-Seq. adata.X is
 normalized by the total counts per cell, scaled to a uniform value, then
 converted to logarithmic space by adding a pseudocount and applying a
 natural log transform, and finally z-scored. This processing was done for
 the MERFISH and snRNA-Seq seperatly before concatenating the two data sets
 together. The raw data was not included in the structure since the units
 would not be equivalent between the two techniques. The obs field contains
 the properties of each observed cell and for this structure are defined as
 follows (this is stored as 'cell_properties' in the csv format)
 -CellType: snRNAseq annotation of cell from snRNAseq analysis alone
 -n_counts: sum of counts within cell -n_genes: number of genes for the
 cell -sample_id: identification of patient (MERFISH measurements) -batch:
 product of anndata concatenation; is unique identifier of technique
 -healthy_annotations: Annotations from analysis of healthy MERFISH data
 alone -leiden_r1.2_joint: leiden resolution used for joint annotations
 -Hep_Type_Joint_1.2: final joint annotation of heaptocytes (based on
 leiden resolution 1.2) -sample_id_sn: identification of patient (snRNAseq
 measurements) ``` ## MERFISH Localizations We also provide the output of
 the MERFISH analysis pipeline after segmentation with baysor. These files
 provide all associated metadata with all identified RNA molecules. These
 files are stored within the rna_metadata folder. Each experiment has a csv
 file associated with the measurement. This file describes the properties
 of each RNA detected through the decoding pipeline (described below). The
 files follow the convention of '[sample id]_[date of
 collection]'. ``` The following fields are included in each csv file:
 -barcode_id: index of gene in codebook -gene_name: name of identified gene
 -fov_id: specific field-of-view (FOV) associated with image in which RNA
 is found. FOV follows sequential order of acquisition -total_magnitude:
 sum of the normalized intensity associated with each pixel of the given
 RNA -abs_position_1: x position of the centroid of the RNA in microns
 -abs_position_2: y position of the centroid of the RNA in microns
 -abs_position_3: z position (depth within the tissue slice) of the
 centroid of the RNA in microns -area: number of pixel assigned to the
 given RNA -error_bit: bit position at which error correction occurred (if
 applicable) -error_dir: direction of error (1 = 1-&gt;0 error (loss of
 signal); 0 = 0-&gt;1 error (gain of signal)) -av_distance: average
 distance to matched barcode -feature_id: cellpose cell id -in_feature:
 True or false indication if RNA in within cellpose cell boundary
 -in_feature_id: cellpose cell id -molecule_id: RNA ID used by baysor
 -prior_segmentation: cell ID used by baysor -confidence: Baysor output
 showing probability of a molecule to be real -cluster: Baysor output
 identifying the molecule cluster -cell: ID of cell assigned cell,
 ""corresponds to noise -assignment_confidence: Baysor output
 indicating confidence of RNA assigned to a correct cell -is_noise: Baysor
 output which indicated if the algorithm should (False) or should not
 (true) be assigned to a cell ```