Processing Your Internal RNAseq Data

1. Overview

This guide will illustrate how you can process your own RNAseq data and utilize it in conjunction with our Eye in a Disk (EiaD) database to conduct differential gene expression analysis.

We will begin by downloading and loading necessary packages for use throughout this tutorial:

Comment out the install.packages if you get errors on the library loads below

# # The following packages will be used throughout this tutorial for data manipulation
# install.packages("tidyverse")
# install.packages("tibble")
# install.packages("data.table")
# install.packages("tximport") 
# Load packages
library(tidyverse)
library(tibble)
library(data.table)
library(tximport)

2. Quick Start

In order to combine your personal RNA-seq dataset with our EiaD database for use in gene expression analysis, it must first be processed using an RNAseq processing tool(kallisto/STAR/etc.). In this tutorial we will be using Salmon. For a a full explanation of how to use Salmon, please visit the latest salmon documentation here.

2.1 Load EiaD Count Data and Metadata

Importing the EiaD gene counts will take some time, as this file is over 175 MB. If you would like to, you can also swap out gene counts for transcript counts, which can be found here. If you utilize the EiaD transcript counts, your local counts will need to be aggregated to the gene level.

# Retrieve metadata and gene counts for EiaD
metadata <- data.table::fread("https://hpc.nih.gov/~mcgaugheyd/eyeIntegration/2023/eyeIntegration23_meta_2023_09_01.built.csv.gz")

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|--------------------------------------------------|
|==================================================|

gene_counts <- data.table::fread("https://hpc.nih.gov/~mcgaugheyd/eyeIntegration/2023/gene_counts.csv.gz")

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We will compare adult human retina tissue against iPSC neuron tissue. The table below shows that ERP126780 (study_accession column) is a possibility for the retina sample. Note how we also save exactly which sample_accessions meet the criteria for our desired sample.

metadata %>% filter(Tissue == 'Retina', Source == 'Native', Age == 'Adult') 

EiaD_retina_samples <- metadata %>% filter(Tissue == 'Retina', Source == 'Native', Age == 'Adult') %>% 
  filter(study_accession == 'ERP126780') %>% 
  pull(sample_accession)

2.2 Process and Load YOUR Personal RNA-seq data

After quantifying our RNAseq dataset using Salmon, we must import our transcript-level estimates and aggregate them to the gene level for gene-level differential expression analysis using tximport. A full guide for the R package can be found here.

You can use the following steps to process and load your personal data:

• Step 1: Use Salmon with the refgenie prebuilt index
• Step 2: Use tximport to load your Salmon data into R

Using SRA Project SRP096788 (non-EiaD iPSC neuron data) for thisexample

Salmon version 1.10.2 was used throughout this guide

The chunk must be run on the (bash) command line (like in “Terminal” on a Mac) NOT in R.

# https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump
fasterq-dump SRR5177944
fasterq-dump SRR5177946
fasterq-dump SRR5177947

Get pre-made reference

This is not the one used in our pipeline, but it is substantially smaller (about 2GB instead of 17GB).

If you still want ours, it is here

wget -O index.tgz http://refgenomes.databio.org/v3/assets/archive/2230c535660fb4774114bfa966a62f823fdb6d21acf138d4/salmon_partial_sa_index?tag=default
tar -xzvf index.tgz
rm index.tgz

salmon quant --index ../default --libType A --seqBias --gcBias --validateMappings \
-p 8 \
SRR5177944.fastq \
-o SRR5177944

salmon quant --index ../default --libType A --seqBias --gcBias --validateMappings \
-p 8 \
SRR5177946.fastq \
-o SRR5177946

salmon quant --index ../default --libType A --seqBias --gcBias --validateMappings \
-p 8 \
SRR5177947.fastq \
-o SRR5177947

Load annotation data and create transcript-to-gene mapping

Since we have counts at the transcript level, we will create a mapping between each transcript and its associated gene so that your locally processed data can be mapped to the gene level:

exon_anno <- rtracklayer::readGFF('https://hpc.nih.gov/~mcgaugheyd/eyeIntegration/2023/2230c535660fb4774114bfa966a62f823fdb6d21acf138d4.gtf.gz')

trying URL 'https://hpc.nih.gov/~mcgaugheyd/eyeIntegration/2023/2230c535660fb4774114bfa966a62f823fdb6d21acf138d4.gtf.gz'
Content type 'application/octet-stream' length 46426050 bytes (44.3 MB)
==================================================
downloaded 44.3 MB

tx2gene <- exon_anno %>% filter(!is.na(transcript_id)) %>% 
  mutate(tx = paste0(transcript_id, '.', transcript_version),
         gene = paste0(gene_id, '.', gene_version)) %>% dplyr::select(tx, gene) %>% unique()

Now, we can use this mapping to load in our Salmon-quantified transcripts:

# import with tximport
txi <- tximport(c('SRR5177944/quant.sf', 'SRR5177946/quant.sf', 'SRR5177947/quant.sf'), 
                type = 'salmon', countsFromAbundance = 'no', txOut = FALSE, tx2gene = tx2gene)

reading in files with read_tsv
1 2 3 
transcripts missing from tx2gene: 8391
summarizing abundance
summarizing counts
summarizing length

Create local metadata

DESeq2 requires metadata for each sample. Some metadata fields we use in EiaD that you can replicate to make your life easier are: - Tissue - Sub_Tissue - Age - Perturbation

# Create metadata for your locally processed samples
local_metadata <- data.frame(sample_accession = c("SRR5177944", "SRR5177946", "SRR5177947"),
                             Tissue = c("Neuron", "Neuron", "Neuron"),
                             Sub_Tissue = c(NA, NA, NA),
                             Age = c(NA, NA, NA),
                             Source = c("iPSC", "iPSC", "iPSC"),
                             Perturbation = c("ALS", NA, NA))

3. DESeq2 Time: Differential Testing

The example above illustrates how to process your RNAseq data using Salmon and load it into R. If your RNAseq data was processed using a tool other than Salmon, please refer to the DESeq2 guide to learn more about how you can load your data into R for use with DESeq2.

Combine EiaD data with the txi object

# match up row names
EiaD_data <- gene_counts[, ..EiaD_retina_samples] %>% as.matrix()
row.names(EiaD_data) <- gene_counts$Gene
# rows to keep
EiaD_keep <- row.names(EiaD_data) %in% row.names(txi$counts)
EiaD_data_subset <- EiaD_data[EiaD_keep, EiaD_retina_samples]

# update txi$counts colnames to something informative
txi$counts <- cbind(txi$counts[EiaD_data_subset %>% row.names,], EiaD_data_subset) %>% as.matrix()
colnames(txi$counts) <- c('SRR5177944', 'SRR5177946', 'SRR5177947', EiaD_retina_samples)

# copy length data of first to match number of samples you add
txi$length <- cbind(txi$length, replicate(ncol(EiaD_data_subset), txi$length[,1]))

# confirm it works by looking at a few rows (if you see NA a problem has happened)
txi$counts %>% as_tibble(rownames = 'ID') %>% sample_n(10)

# Build combined DESeq counts matrix for EiaD and locally processed samples
DESeq_counts <- txi$counts

Combine EiaD metadata with local metadata

# Pull EiaD metadata
EiaD_metadata <- metadata %>% filter(sample_accession %in% c(EiaD_retina_samples))
# Combine metadata with the metadata we created above for our locally processed iPSC neuron samples
DESeq_meta <- bind_rows(local_metadata, EiaD_metadata)

Run DESeq2

# install.packages("DESeq2")
library(DESeq2)

Loading required package: S4Vectors
Loading required package: stats4
Loading required package: BiocGenerics

Attaching package: ‘BiocGenerics’

The following object is masked from ‘package:limma’:

    plotMA

The following objects are masked from ‘package:lubridate’:

    intersect, setdiff, union

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    combine, intersect, setdiff, union

The following objects are masked from ‘package:stats’:

    IQR, mad, sd, var, xtabs

The following objects are masked from ‘package:base’:

    anyDuplicated, append, as.data.frame, basename, cbind, colnames, dirname, do.call, duplicated, eval, evalq,
    Filter, Find, get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget, order, paste, pmax,
    pmax.int, pmin, pmin.int, Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply, union,
    unique, unsplit, which.max, which.min


Attaching package: ‘S4Vectors’

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Loading required package: IRanges

Attaching package: ‘IRanges’

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Loading required package: GenomicRanges
Loading required package: GenomeInfoDb
Loading required package: SummarizedExperiment
Loading required package: MatrixGenerics
Loading required package: matrixStats

Attaching package: ‘matrixStats’

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Attaching package: ‘MatrixGenerics’

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    colCumsums, colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs, colMads, colMaxs, colMeans2, colMedians,
    colMins, colOrderStats, colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds, colSums2, colTabulates,
    colVarDiffs, colVars, colWeightedMads, colWeightedMeans, colWeightedMedians, colWeightedSds, colWeightedVars,
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    rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps, rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians,
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    rowVarDiffs, rowVars, rowWeightedMads, rowWeightedMeans, rowWeightedMedians, rowWeightedSds, rowWeightedVars

Loading required package: Biobase
Welcome to Bioconductor

    Vignettes contain introductory material; view with 'browseVignettes()'. To cite Bioconductor, see
    'citation("Biobase")', and for packages 'citation("pkgname")'.


Attaching package: ‘Biobase’

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# build dds
dds_EiaD_iPSC_neuron <-  DESeqDataSetFromMatrix(countData = round(DESeq_counts),
                                                colData = DESeq_meta,
                                                design = ~ Tissue)

converting counts to integer mode

# build deseq2 table
DESeq2Table <- DESeq(dds_EiaD_iPSC_neuron)

estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
-- replacing outliers and refitting for 169 genes
-- DESeq argument 'minReplicatesForReplace' = 7 
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing

Top 100 differentially expressed genes

top100 <- results(DESeq2Table) %>% as_tibble(rownames = 'ensembl_gene_id') %>% arrange(padj) %>% head(100)
top100

Now human readable

We see how some of the top positive and negative log2FoldChange genes are associated with Retina and iPSC Motor Neurons, respectively.

You can click on the column fields to sort by that column.

Diff results

results(DESeq2Table) %>% 
  as_tibble(rownames = 'ENSEMBL') %>% 
  arrange(padj) %>% head(10) %>% 
  mutate(ENSEMBL = gsub('\\.\\d+','',ENSEMBL)) %>% 
  left_join(conv_table, by = 'ENSEMBL') %>% 
  relocate(hgnc_symbol) %>% 
  arrange(log2FoldChange) %>% 
  DT::datatable()

Error in `relocate()`:
! Can't subset columns that don't exist.
✖ Column `hgnc_symbol` doesn't exist.
Backtrace:
 1. ... %>% DT::datatable()
 6. dplyr:::relocate.data.frame(., hgnc_symbol)

PCA to look for patterns across samples

We see that PC1 separates retina and iPSC neuron tissue.

vst <- varianceStabilizingTransformation(DESeq2Table)
library(matrixStats)
ntop = 1000
Pvars <- rowVars(assay(vst))
select <- order(Pvars, decreasing = TRUE)[seq_len(min(ntop, 
                                                      length(Pvars)))]
PCA <- prcomp(t(assay(vst)[select, ]), scale = F)
percentVar <- round(100*PCA$sdev^2/sum(PCA$sdev^2),1)
dataGG = data.frame(PC1 = PCA$x[,1], PC2 = PCA$x[,2], 
                    PC3 = PCA$x[,3], PC4 = PCA$x[,4], 
                    Tissue = colData(vst)$Tissue,
                    Source=colData(vst)$Source)
ggplot(dataGG, aes(PC1, PC2, color=Tissue, shape=Source)) +
  geom_point(size=3) +
  xlab(paste0("PC1: ",percentVar[1],"% variance")) +
  ylab(paste0("PC2: ",percentVar[2],"% variance")) + 
  cowplot::theme_cowplot()

4. Conclusions

1. We show how to pull the EiaD gene counts and metadata
2. We show how to process your local RNAseq datasets and create metadata for use in DESeq2
3. We show how to combine EiaD with your locally processed data for use in DESeq2
4. We do a very brief analysis in DESeq2