<p>The solution was to use a different library as I expected. It should be <code>library(TxDb.Hsapiens.UCSC.hg19.ensGene)</code>. And also, as specified in the question, to remove everything after the dot in the <code>Name</code> column of the <code>quant.sf</code> files.</p>

CC BY-SA 3.0
2018-04-11T21:09:08.857Z
2018-04-11T21:09:08.857Z
Nikita Vlasenko
<p>Do your ids in the expression file (from gencode) match up with the gene IDs in the transcript file (from UCSC?) I'm willing to bet that they don't. Stay consistent throughout your process. Either pull down the gencode transcript to gene translations, or do your pseudoalignments to UCSC transcripts.</p>

CC BY-SA 3.0
2018-04-10T21:56:39.240Z
I already got the `salmon` aligned data, that was aligned to the `gencode` file. Now I do not know what `TxDb` library file I need to use.
Nikita Vlasenko
CC BY-SA 3.0
2018-04-11T10:48:02.547Z
see answer [here](https://support.bioconductor.org/p/55509)
heathobrien
CC BY-SA 3.0
2018-04-10T21:30:54.183Z
2018-04-10T21:30:54.183Z
chrisamiller
<p>I am trying to use <code>tximport</code> to get raw count matrices of a bulk RNA-seq sequencing experiment. I followed the tutorial:</p>

<p><a href="https://bioconductor.org/packages/3.7/bioc/vignettes/tximport/inst/doc/tximport.html" rel="nofollow noreferrer">https://bioconductor.org/packages/3.7/bioc/vignettes/tximport/inst/doc/tximport.html</a></p>

<p>Where they generate <code>tx2gene</code> in the following way:</p>

<pre><code>library(TxDb.Hsapiens.UCSC.hg19.knownGene)
txdb &lt;- TxDb.Hsapiens.UCSC.hg19.knownGene
k &lt;- keys(txdb, keytype = "TXNAME")
tx2gene &lt;- select(txdb, k, "GENEID", "TXNAME")
</code></pre>

<p>So, I reasoned to do it the same way. But since my data is mouse one, I used a different gene annotation file:</p>

<pre><code>library(TxDb.Mmusculus.UCSC.mm10.knownGene)
txdb &lt;- TxDb.Mmusculus.UCSC.mm10.knownGene
k &lt;- keys(txdb, keytype = "TXNAME")
tx2gene &lt;- select(txdb, k, "GENEID", "TXNAME")
</code></pre>

<p>The transcript annotation file that I used when running <code>salmon</code> is <code>gencode.vM13.transcripts.fa.sidx</code>. Then when I try getting <code>txi</code> object:</p>

<pre><code>txi &lt;- tximport(files, type = "salmon", tx2gene = tx2gene)
</code></pre>

<p>It raises the error:</p>

<p><a href="https://i.sstatic.net/vCt2U.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/vCt2U.png" alt="enter image description here"></a></p>

<p>Seems like I am creating <code>tx2gene</code> in the wrong way. Maybe I need to use a different library, not <code>TxDb.Mmusculus.UCSC.mm10.knownGene</code>. Any suggestions would be greatly appreciated.</p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>I looked into the <code>quant.sf</code> files that I got from <code>salmon</code> and it looked the following way:</p>

<p><a href="https://i.sstatic.net/LmbuM.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/LmbuM.png" alt="enter image description here"></a></p>

<p>A colleague adviced me to strip away everything that follows the dot in the <code>Name</code> column, so it started to look:</p>

<p><a href="https://i.sstatic.net/LaSa2.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/LaSa2.png" alt="enter image description here"></a></p>

<p>However, still no luck. The identifiers in the <code>k &lt;- keys(txdb, keytype = "TXNAME")</code> look like that:</p>

<pre><code>"uc007afg.1" "uc007afh.1" "uc007afi.2" "uc011wht.1" "uc011whu.1" 
"uc057aty.1"
</code></pre>

<p>I suppose I need to convert these identifiers first to <code>ENS</code>-like ones, but do not know how to.</p>

<rna-seq><salmon><tximport><tx2gene>
tx2gene file for tximport issue
# tx2gene file for tximport issue

(Asked by: username_0 on 2018-04-10)

I am trying to use tximport to get raw count matrices of a bulk RNA-seq sequencing experiment. I followed the tutorial:
https://bioconductor.org/packages/3.7/bioc/vignettes/tximport/inst/doc/tximport.html
Where they generate tx2gene in the following way:
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
k <- keys(txdb, keytype = "TXNAME")
tx2gene <- select(txdb, k, "GENEID", "TXNAME")

So, I reasoned to do it the same way. But since my data is mouse one, I used a different gene annotation file:
library(TxDb.Mmusculus.UCSC.mm10.knownGene)
txdb <- TxDb.Mmusculus.UCSC.mm10.knownGene
k <- keys(txdb, keytype = "TXNAME")
tx2gene <- select(txdb, k, "GENEID", "TXNAME")

The transcript annotation file that I used when running salmon is gencode.vM13.transcripts.fa.sidx. Then when I try getting txi object:
txi <- tximport(files, type = "salmon", tx2gene = tx2gene)

It raises the error:

Seems like I am creating tx2gene in the wrong way. Maybe I need to use a different library, not TxDb.Mmusculus.UCSC.mm10.knownGene. Any suggestions would be greatly appreciated.

Update

I looked into the quant.sf files that I got from salmon and it looked the following way:

A colleague adviced me to strip away everything that follows the dot in the Name column, so it started to look:

However, still no luck. The identifiers in the k <- keys(txdb, keytype = "TXNAME") look like that:
"uc007afg.1" "uc007afh.1" "uc007afi.2" "uc011wht.1" "uc011whu.1" 
"uc057aty.1"

I suppose I need to convert these identifiers first to ENS-like ones, but do not know how to.

**Tags:** rna-seq, salmon, tximport, tx2gene

---

## 2 answers

### Answer by username_0 on 2018-04-11. Score: 2 | Accepted answer

The solution was to use a different library as I expected. It should be `library(TxDb.Hsapiens.UCSC.hg19.ensGene)`. And also, as specified in the question, to remove everything after the dot in the `Name` column of the `quant.sf` files.


---

### Answer by username_1 on 2018-04-10. Score: 1

Do your ids in the expression file (from gencode) match up with the gene IDs in the transcript file (from UCSC?) I'm willing to bet that they don't. Stay consistent throughout your process. Either pull down the gencode transcript to gene translations, or do your pseudoalignments to UCSC transcripts.

> **Comments:**
> **username_0**: I already got the `salmon` aligned data, that was aligned to the `gencode` file. Now I do not know what `TxDb` library file I need to use.  
> **username_2**: see answer [here](https://support.bioconductor.org/p/55509)  

---

