I'm quite new to RNA-sequencing and am playing around with data to get a handle on it. I have quantified with Kallisto and am using tximport to summarize transcript counts for differential gene expression analysis.

I am running into a problem associating gene ID's with my transcripts for the summarization portion. I believe that the likely cause is the actual TxDb library I am using and that it may be different from the transcriptome file I used, but I am not sure and my attempts at solving this haven't been successful.

I am working with human samples. I quantified my transcripts using this transcriptome file for homo sapiens. I have 6 samples, 3 WT replicates, and 3 KO replicates.

1. I created a vector pointing to my kallisto files as detailed in the tximport manual.

   files <- file.path(dir, "kallisto", samples$run, "abundance.tsv")

2. I created a data.frame from a TxDb object to construct the tx2gene table.

   library(TxDb.Hsapiens.UCSC.hg38.knownGene)

   txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene

   k <- keys(txdb, keytype = "GENEID")

   df <- select(txdb, keys = k, keytype = "GENEID", columns = "TXNAME")

   tx2gene <- df[, 2:1] # tx ID, then gene ID

But head(tx2gene) produces:

TXNAME GENEID
1 uc002qsd.4      1
2 uc002qsf.2      1
3 uc003wyw.1     10
4 uc002xmj.3    100
5 uc010xbn.1   1000
6 uc002kwg.2   1000

This obviously isn't right.

1. Using tximport's tximport function.

   library(tximport)

   library(readr)

   txi <- tximport(files, type = "kallisto", tx2gene = tx2gene, reader = read_tsv)

   names(txi)

Does the following:

txi $abundance

sample 1 sample 2 sample 3 sample 4 sample 5 sample 6

$counts

sample 1 sample 2 sample 3 sample 4 sample 5 sample 6

$length

sample 1 sample 2 sample 3 sample 4 sample 5 sample 6

$countsFromAbundance

[1] "no"

and head(txi$counts):

head(txi$counts)

sample 1 sample 2 sample 3 sample 4 sample 5 sample 6

I'm not completely sure what i'm doing incorrectly. I'll give it another shot after lunch, it might just be the frustration at this point but any help is appreciated.