Calibrating RNA-Seq Data with Drosophila Spike-ins

Cited from External calibration with Drosophila whole-cell spike-ins delivers absolute mRNA fold changes from human RNA-Seq and qPCR data

Calibrating RNA-Seq data with Drosophila spike-ins

1. Concatenate the human and Drosophila genome (alternatively, concatenate the transcriptomes).
2. Generate a genome (or transcriptome) index for your preferred alignment or counting algorithm if necessary.
3. Align the reads to the concatenated genome/transcriptome.
4. Generate read counts per gene/transcript/exon.
5. Split the count table into two, one containing human genes, the other Drosophila genes. If you use DESeq2, you can also read in the complete count table and subset the resulting "DESeqDataSet" data object.
6. Calculate sample scaling factors from the Drosophila read counts. When using DESeq2, use the function “sizeFactors” on the “DESeqDataSet” containing the Drosophila genes/transcripts/exons.
7. Apply the size factors to the human read counts by dividing the counts of each individual gene of one sample by the respective sample size factor. When using DESeq2, overwrite the size factor slot of the human data object with the size factors estimated from the Drosophila data (pseudo-code: 'sizeFactors(HumanData) ← sizeFactors(DrosiData)').

Overview in External calibration with whole-cell spike-ins delivers absolute mRNA fold changes