Associate annotation information - bg_ids NULL

[Macdot3]

Hi Federico, thanks for your app which can really make my work a lot easier! For the first time I am approaching RNAseq data analysis and I am adapting my data to the documentation workflow. Once I get to the re_enrich part, when I create bg_ids it returns me a NULL value. How can I solve this problem? I tried adding the annotation to the dds object but I get no results. I'll write to you below my code.

colData <- read.table('DATI_GeneTonic/samples_table.txt', sep='\t', row.names = 1,header = T) files <- file.path('DATI_GeneTonic', colData$seqname_quant, "quant.sf") names(files) <- rownames(colData) tx2gene <- readRDS("DATI_GeneTonic/tx2gene.gencode.v38.annotation.RDS") head(tx2gene)

txi <- tximport(files, type="salmon", tx2gene=tx2gene) ddsTxi <- DESeqDataSetFromTximport(txi, colData = colData, design = ~kit+condition) dds <- ddsTxi dds$symbol <- mapIds(org.Hs.eg.db, keys=gene_ids, column="SYMBOL", keytype="ENSEMBL") rownames(dds) <- substr(rownames(dds), 1, 15) keep <- rowSums(counts(dds)) > 0 dds <- dds[keep,] dds <- DESeq(dds) res_control_mutant <- results(dds, contrast = c("condition", "control", "mutant"), lfcThreshold = 1, alpha = 0.05) res_control_mutant$symbol <- mapIds(org.Hs.eg.db, keys=ens.str, column="SYMBOL", keytype="ENSEMBL", multiVals="first") de_symbols_control_mutant <- deseqresult2df(res_control_mutant, FDR = 0.05)$symbol

bg_ids <- rowData(dds)$symbol[rowSums(counts(dds)) > 0] NULL

Thanks Marco

[federicomarini]

Hi Marco, this is a bit odd. Can you check a bit more the content of your dds object? For example, what is the printout of

dds
rowData(dds)

The important part is to select the background properly, and you are on the right way to do so. Feel free to reach out via email if you need more support, although this is not strictly related to GeneTonic itself. Federico

[Macdot3]

This is my printout:

> dds
class: DESeqDataSet 
dim: 60230 18 
metadata(1): version
assays(2): counts avgTxLength
rownames(60230): ENSG00000000003.15 ENSG00000000005.6 ... ENSG00000288724.1 ENSG00000288725.1
rowData names(0):
colnames(18): FB_bruda_ckt_10x FB_bruda_ckt_1x ... FB_zamac_ckt_1x FB_zamac_nt
colData names(10): seqname name ... subtype seqname_quant
> rowData(dds)
DataFrame with 60230 rows and 0 columns