---
title: "RNA-seq quantifications from GEO"
author: "Sean Davis"
format:
  html:
    toc: true
vignette: >
  %\VignetteIndexEntry{RNA-seq quantifications from GEO}
  %\VignetteEngine{quarto::html}
  %\VignetteEncoding{UTF-8}
---

A major barrier to reusing public RNA-seq data has been that every submitter
processes raw reads differently — different aligners, references, and counting
rules — so counts from two studies are rarely directly comparable. To address
this, NCBI computes a **uniform set of RNA-seq quantifications** for many GEO
Series using a single consistent pipeline. This article explains what those are
and how GEOquery retrieves them.


``` r
library(GEOquery)
```

## NCBI-computed quantifications vs. submitter files

There are two distinct things you might mean by "the counts" for a GEO RNA-seq
study:

1. **Submitter-provided processed files** — whatever the authors uploaded as
   supplementary files. Formats and gene models vary by study; GEOquery lists and
   downloads them (`getGEOSuppFiles()`) but does not interpret them.
2. **NCBI-computed quantifications** — a raw-count matrix (and accompanying
   annotation) that NCBI generated uniformly from the study's SRA runs. These are
   comparable *across* studies and are what the GEOquery RNA-seq functions
   target.

Not every Series has NCBI quantifications; you can check first.

## Checking availability and loading


``` r
# Does this Series have NCBI-computed quantifications?
hasRNASeqQuantifications("GSE164073")
#> [1] TRUE
```


``` r
# Load them as a SummarizedExperiment
se <- getRNASeqData("GSE164073")
se
#> class: SummarizedExperiment 
#> dim: 39376 18 
#> metadata(5): experimentData annotation protocolData genomeInfo
#>   created_at
#> assays(1): counts
#> rownames(39376): 100287102 653635 ... 4576 4571
#> rowData names(18): GeneID Symbol ... GOProcess GOComponent
#> colnames(18): GSM4996084 GSM4996085 ... GSM4996100 GSM4996101
#> colData names(45): title geo_accession ... time.point.ch1 tissue.ch1
```

The result is a `SummarizedExperiment`: `assay(se)` is the raw count matrix,
`rowData(se)` carries gene annotation, and `colData(se)` the sample metadata.
Because it is a `SummarizedExperiment`, it drops straight into the differential
expression workflows described in the
[downstream analysis](downstream-analysis.html) article.


``` r
assay(se)[1:5, 1:3]
#>           GSM4996084 GSM4996085 GSM4996086
#> 100287102          2          5          3
#> 653635           244        236        337
#> 102466751         25         17         34
#> 107985730          1          1          1
#> 100302278          0          0          0
```

## Why raw counts (and what to do with them)

The NCBI quantifications are deliberately **raw counts**, not normalized values,
because the correct normalization depends on your downstream method. Count-based
differential expression tools expect raw counts and model the counting noise
themselves:

- [DESeq2](https://bioconductor.org/packages/DESeq2)
- [edgeR](https://bioconductor.org/packages/edgeR)
- [limma](https://bioconductor.org/packages/limma) with `voom`

See [downstream analysis](downstream-analysis.html) for a worked sketch.

## When you need the submitter's files instead

If a study has no NCBI quantifications, or you specifically need the authors'
processed output (e.g. TPM, or a custom gene model), fall back to the
supplementary files and read them yourself:


``` r
getGEOSuppFiles("GSE164073", fetch_files = FALSE)   # inspect first
#>                               fname
#> 1 GSE164073_Eye_count_matrix.csv.gz
#>                                                                                                   url
#> 1 https://ftp.ncbi.nlm.nih.gov/geo/series/GSE164nnn/GSE164073/suppl/GSE164073_Eye_count_matrix.csv.gz
```

## Where to go next

- [Finding and downloading data](finding-and-downloading-data.html) — search for
  RNA-seq studies and cache large downloads.
- [From GEO to downstream analysis](downstream-analysis.html) — DESeq2 / edgeR /
  limma-voom from a count `SummarizedExperiment`.