Exploring SpaceRanger summary metrics

LIBD Rstats Club: Exploring SpaceRanger summary metrics
Presented By : Manisha Barse
Date: November 01, 2024
Recording link to our rstats club presentation: https://youtu.be/cJqsbDh0ZtI

An overview of metrics summary CSV files generated by SpaceRanger when processing Visium spatially-resolved transcriptomics.

Transcript

1. Exploring SpaceRanger summary metrics Manisha Barse 2024/11/01
2. Objectives What is spaceranger? What is a spaceranger web summary file by 10x genomics? What are the components to it? What are the spaceranger summary_metrics.csv? What are the most used metrics? What data are we looking at? Compare DLPFC_ASD, spatialDLPFC, and Human Pilot Plots
3. What is SpaceRanger? Space Ranger: set of analysis pipelines for processing 10x Genomics Visium sequence data (FASTQ files) with high resolution microscope images of tissue- brightfield and fluorescence. SpaceRanger allows to map whole transcriptome in formalin fixed paraffin embedded (FFPE) and fresh frozen tissues. Provides pipelines for end to end analysis of Visium Spatial Gene Expression experiments. https://lmweber.org/Visium-data-preprocessing/space-ranger.html https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/what-is-space-ranger
4. Visium Slides • V1: Visium Spatial Gene Expression Slide (v1, 6.5 mm) • V4: Visium CytAssist Spatial Gene Expression Slide (v2, 6.5 mm) • V5: Visium CytAssist Spatial Gene Expression Slide (v2, 11 mm) • H1: Visium HD Slide (6.5 mm) • A1, B1, C1, D1 for Visium Spatial Gene Expression slides, v1, 6.5 mm • A, B for Visium CytAssist Spatial Gene Expression slide, v2, 11 mm • A1, D1 for Visium CytAssist Spatial Gene Expression slides, v2, 6.5 mm, and Visium HD slides, 6.5 mm
5. SpaceRanger Outputs Pipeline: spaceranger count Visium CytAssist • web_summary.html • tissue_positions.csv: spot coordinates • tissue_lowres_image.png : max 600 pixels • tissue_lowres_image.png : max 2000 pixels • raw_features_bc_matrix • SpaceRanger v2.1 and later - support reference-free spot deconvolution function for v1/v2, not for visium HD Output files overview Feature barcode matrices that contains both tissue-associated as well as background barcodes and are used in secondary analysis in R/Python Data summary with images, metrics, and plots that can be used for quality assessment as well as errors and warnings related to data quality
6. Web Summary file First file to look after running SpaceRanger - tells if the run was successful.
7. Web Summary file (cont.) visium HD example(different tabs) link All summaries together link SpaceRanger versions Terms link
8. metrics_summary.csv metrics_summary.csv file, which includes the metrics displayed in the interactive website web_summary.html. Note: format of this file has changed across spaceranger versions. Terms: link Probe based: link ref
9. Sample.ID, Number.of.Spots.Under.Tissue Number.of.Reads Mean.Reads.per.Spot Mean.Reads.Under.Tissue.per.Spot Fraction.of.Spots.Under.Tissue Valid.Barcodes Valid.UMIs Sequencing.Saturation Q30.Bases.in.Barcode Q30.Bases.in.UMI Fraction.Reads.in.Spots.Under.Tissue Median.Genes.per.Spot Median.UMI.Counts.per.Spot Q30.Bases.in.Probe.Read Reads.Mapped.to.Probe.Set Reads.Mapped.Confidently.to.Probe.Set Genes.Detected Reads.Mapped.Confidently.to.the.Filtered. Probe.Set Number.of.Genes Number.of.Genes....10.UMIs Reads.Half.Mapped.to.Probe.Set Reads.Split.Mapped.to.Probe.Set Estimated.UMIs.from.Genomic.DNA Estimated.UMIs.from.Genomic.DNA.per.Un spliced.Probe Q30.Bases.in.RNA.Read Reads.Mapped.to.Genome Reads.Mapped.Confidently.to.Genome Reads.Mapped.Confidently.to.Intergenic.Regions Reads.Mapped.Confidently.to.Intronic.Regions Reads.Mapped.Confidently.to.Exonic.Regions Reads.Mapped.Confidently.to.Transcriptome Reads.Mapped.Antisense.to.Gene Total.Genes.Detected Probe based CytAssist Data Direct placement Visium Data
10. What data are we looking at Human Pilot: n= 3 NTC (2 pairs each) spatialDLPFC: n = 10 NTC(3 blocks: ant, mid, post) DLPFC_ASD: n = 16 (8 NTC, 8 ASD) *NTC: neurotypical control *ASD: Autism Spectrum Disorder
11. Plots https://github.com/manishabarse/spaceranger_plots We observe: • High UMI counts, mapping confidence in Probe Based CytAssist
12. Acknowledgements Leonardo Collado-Torres Cynthia S Cardinault Louise Huuki Nick Eagles Kristen Maynard Stephanie Page Ryan Miller
13. Thank You!