Hello everyone,
Hello Everyone,
I am having issue with when trying to aggregate transcript abundances to the gene level (when txOut=FALSE) but it works fine with txOut=TRUE.
Here are the steps I followed:
- Produced bam file using Gencode transcript fasta file.
- Further sorted and index them.
- Used Nanocount to produce abundance.tsv file.
- Created gencode dataframe using gencode gtf file containing only two columns
target_idandgene_name - Read quant file using path command
Here is the input tsv files:
read_tsv(path)
target_id raw est_count tpm transcript_length
<chr> <dbl> <dbl> <dbl> <dbl>
1 ENST00000362079.2 0.0109 4264. 10883. 784
2 ENST00000343262.9 0.0102 3985. 10172. 945
3 ENST00000567815.5 0.00993 3891. 9931. 712
4 ENST00000501597.3 0.00902 3533. 9018. 469
5 ENST00000651669.1 0.00826 3235. 8257. 352
6 ENST00000361624.2 0.00790 3096. 7902. 1542
7 ENST00000270625.7 0.00782 3066. 7825. 573
8 ENST00000222247.10 0.00779 3051. 7788. 634
9 ENST00000229239.10 0.00753 2949. 7526. 1285
10 ENST00000530721.5 0.00744 2917. 7445. 796
Here is the tx file for aggregating transcript abundances to the gene level
tx_gencode_gtf_local
# A tibble: 3,424,907 × 2
target_id gene_name
<chr> <chr>
1 NA DDX11L2
2 ENST00000456328.2 DDX11L2
3 ENST00000456328.2 DDX11L2
4 ENST00000456328.2 DDX11L2
5 ENST00000456328.2 DDX11L2
6 NA DDX11L1
7 ENST00000450305.2 DDX11L1
8 ENST00000450305.2 DDX11L1
9 ENST00000450305.2 DDX11L1
10 ENST00000450305.2 DDX11L1
when I am running this command:
Txi_gene <- tximport(path,
type = "none",
tx2gene = tx_gencode_gtf_local,
txIdCol = "transcript_id",
abundanceCol = "tpm",
countsCol = "raw",
lengthCol = "transcript_length",
countsFromAbundance = "lengthScaledTPM",
txOut = FALSE,
ignoreTxVersion = TRUE)
I am getting error:
Error in .local(object, ...) :
None of the transcripts in the quantification files are present
in the first column of tx2gene. Check to see that you are using
the same annotation for both.
Example IDs (file): [, ...]
Example IDs (tx2gene): [ENST00000456328.2, ENST00000450305.2, ENST00000488147.1, ...]
This can sometimes (not always) be fixed using 'ignoreTxVersion' or 'ignoreAfterBar'.
when I tried txIdCol = "target_id", it showed error with txOut=TRUE so I tried with txIdCol = "transcript_id". But it did not work. Also I tried with ignoreTxVersion=FALSE when tx_gencode_gtf_local ENST_ID does not have version information, but it failed with same error.
Thank you
Mohit
1 answer
These arguments
txIdCol = "transcript_id",
abundanceCol = "tpm",
countsCol = "raw",
lengthCol = "transcript_length",
have to be correct names of columns of the quantification files.
I would also remove NA from tx2gene table. I don't think it matters but just in case.
You don't need to use ignoreTxVersion=TRUE unless you have mismatched version information across quantification file and tx2gene (that is, the quantification file has versioned transcripts but the tx2gene does not).
You probably want txOut=FALSE (I assume, since you are bothering providing a tx2gene table).
Here are the steps I followed but it is giving error:
gtf <- rtracklayer::import('gencode.v43.annotation.gtf')
gtf_df=as.data.frame(gtf)
tx_gencode_gtf <- dplyr::select(gtf_df, "transcript_id", "gene_name")
tx_gencode_gtf <- dplyr::rename(tx_gencode_gtf,target_id = transcript_id)
tx_gencode_gtf <- as_tibble(tx_gencode_gtf)
clean_tx_gencode_gtf <- na.omit(tx_gencode_gtf)
clean_tx_gencode_gtf_unique <- clean_tx_gencode_gtf %>%
distinct(target_id, .keep_all = TRUE)
Followed by:
Txi_gene <- tximport(
path,
type = "none",
tx2gene = "clean_tx_gencode_gtf_unique",
txOut = FALSE,
countsFromAbundance = "lengthScaledTPM",
txIdCol = "transcript_name", # Spcify the correct column name for transcript IDs
abundanceCol = "tpm",
countsCol = "est_count",
lengthCol = "transcript_length",
ignoreTxVersion = FALSE,
ignoreAfterBar = TRUE,
importer = read_tsv
)
Error:
chr (1): transcript_name
dbl (4): raw, est_count, tpm, transcript_length
Error in tximport(path, type = "none", tx2gene = "clean_tx_gencode_gtf_unique", :
all(txId == raw[[txIdCol]]) is not TRUE
Note: I tried with both clean_tx_gencode_gtf and clean_tx_gencode_gtf_unique. But it is giving the same error. abundance.tsv files are generated using similar method.
Here is the structure of abundance.tsv file:
Yes. Earlier I have removed anything after | and used transcript ids for aggregating them to gene level but it was not working. Now, with above mentioned transcript name, I used ignoreAfterBar = TRUE, to avoid using anything after |. But as you can see it was giving error. I will remove everything after | in excel and change txIdCol to "target_id" and txOut to FALSE and test it again.
Don't use Excel. It's a bad tool for processing large plain text content. Use sed/awk instead.
If ignoreAfterBar takes care of the messy data in the column, the error probably happens because there's some transcript ID that is only present in the GTF file (tx2gene object) or the input files. Check if that's the case.
Here is my updated abdundance.tsv files:
transcript_name raw est_count tpm transcript_length
<chr> <dbl> <dbl> <dbl> <dbl>
1 ENST00000362079.2 0.0109 4264. 10883. 784
2 ENST00000343262.9 0.0102 3985. 10172. 945
3 ENST00000567815.5 0.00993 3891. 9931. 712
4 ENST00000501597.3 0.00902 3533. 9018. 469
5 ENST00000651669.1 0.00826 3235. 8257. 352
6 ENST00000361624.2 0.00790 3096. 7902. 1542
7 ENST00000270625.7 0.00782 3066. 7825. 573
8 ENST00000222247.10 0.00779 3051. 7788. 634
9 ENST00000229239.10 0.00753 2949. 7526. 1285
10 ENST00000530721.5 0.00744 2917. 7445. 796
Here is the two column data.frame that has transcript IDs in the first column and gene names
clean_tx_gencode_gtf_unique
# A tibble: 252,913 × 2
target_id gene_name
<chr> <chr>
1 ENST00000456328.2 DDX11L2
2 ENST00000450305.2 DDX11L1
3 ENST00000488147.1 WASH7P
4 ENST00000619216.1 MIR6859-1
5 ENST00000473358.1 MIR1302-2HG
6 ENST00000469289.1 MIR1302-2HG
7 ENST00000607096.1 MIR1302-2
8 ENST00000417324.1 FAM138A
9 ENST00000461467.1 FAM138A
10 ENST00000606857.1 OR4G4P
tximport:
Txi_gene <- tximport(
path,
type = "none",
tx2gene = "clean_tx_gencode_gtf_unique",
txOut = FALSE,
countsFromAbundance = "lengthScaledTPM",
txIdCol = "target_id", # Spcify the correct column name for transcript IDs
abundanceCol = "tpm",
countsCol = "est_count",
lengthCol = "transcript_length",
ignoreTxVersion = FALSE,
importer = read_tsv
)
It is giving error
Error in `colnames<-`(`*tmp*`, value = c("tx", "gene")) :
attempt to set 'colnames' on an object with less than two dimensions
Hello Ram,
I tried all possible things but it is still giving error:
When
Txi_gene <- tximport(
path,
type = "none",
tx2gene = "clean_tx_gencode_gtf",
txOut = FALSE,
countsFromAbundance = "lengthScaledTPM",
txIdCol = "target_id", # Spcify the correct column name for transcript IDs
abundanceCol = "tpm",
countsCol = "est_count",
lengthCol = "transcript_length",
ignoreTxVersion = FALSE,
importer = read_tsv
)
It is giving error:
Error in `colnames<-`(`*tmp*`, value = c("tx", "gene")) :
attempt to set 'colnames' on an object with less than two dimensions
and when I am using
Txi_gene <- tximport(
path,
type = "none",
tx2gene = clean_tx_gencode_gtf,
txOut = FALSE,
countsFromAbundance = "lengthScaledTPM",
txIdCol = "target_id", # Spcify the correct column name for transcript IDs
abundanceCol = "tpm",
countsCol = "est_count",
lengthCol = "transcript_length",
ignoreTxVersion = TRUE,
importer = read_tsv
)
removing duplicated transcript rows from tx2gene
Error in .local(object, ...) :
None of the transcripts in the quantification files are present
in the first column of tx2gene. Check to see that you are using
the same annotation for both.
Example IDs (file): [, ...]
Example IDs (tx2gene): [ENST00000456328.2, ENST00000450305.2, ENST00000488147.1, ...]
This can sometimes (not always) be fixed using 'ignoreTxVersion' or 'ignoreAfterBar'.
Thank you
Mohit
Hello Josie,
Here is the tsv file:
A tibble: 1,010,956 × 5
target_id raw est_count tpm transcript_length
<chr> <dbl> <dbl> <dbl> <dbl>
1 ENST00000362079.2 0.0109 4264. 10883. 784
2 ENST00000343262.9 0.0102 3985. 10172. 945
3 ENST00000567815.5 0.00993 3891. 9931. 712
4 ENST00000501597.3 0.00902 3533. 9018. 469
5 ENST00000651669.1 0.00826 3235. 8257. 352
6 ENST00000361624.2 0.00790 3096. 7902. 1542
Here is tx2gene dataframe having two columns Target_id and Gene_name tx_gencode_gtf_local A tibble: 252,913 × 2
target_id gene_name
<chr> <chr>
1 ENST00000362079.2 MT-CO3
2 ENST00000361739.1 MT-CO2
3 ENST00000387347.2 MT-RNR2
4 ENST00000343262.9 RPS2
5 ENST00000229239.10 GAPDH
6 ENST00000361624.2 MT-CO1
7 ENST00000501597.3 RPL41
8 ENST00000567815.5 RPL13
9 ENST00000651669.1 RPS27
10 ENST00000361899.2 MT-ATP6
when I am trying to run tximport with only est_count (commented out #countsFromAbundance, abundanceCol, and lengthCol) , it is giving error
Error in c(abundanceCol, countsCol, lengthCol) in names(raw) : argument "abundanceCol" is missing, with no default
Txi_gene <- tximport(
+ path,
+ type = "none",
+ tx2gene = "tx_gencode_gtf_local",
+ txOut = FALSE,
+ #countsFromAbundance = "lengthScaledTPM",
+ txIdCol = "target_id", # Spcify the correct column name for transcript IDs
+ #abundanceCol = "tpm",
+ countsCol = "est_count",
+ #lengthCol = "transcript_length",
+ ignoreTxVersion = FALSE,
+ #ignoreAfterBar = TRUE,
+ importer = read_tsv
+ )
Rows: 252739 Columns: 5
Column specification
Delimiter: "\t"
chr (1): transcript_id
dbl (4): raw, est_count, tpm, transcript_length
Use spec() to retrieve the full column specification for this data.
Specify the column types or set `show_col_types = FALSE` to quiet this message.
Error in c(abundanceCol, countsCol, lengthCol) in names(raw) :
argument "abundanceCol" is missing, with no default
Did you try the last thing I mentioned:
Txi_gene <- tximport(
path,
type = "none",
tx2gene = clean_tx_gencode_gtf,
txOut = FALSE,
countsFromAbundance = "lengthScaledTPM",
txIdCol = "target_id", # Spcify the correct column name for transcript IDs
abundanceCol = "tpm",
countsCol = "est_count",
lengthCol = "transcript_length",
ignoreTxVersion = TRUE,
ignoreAfterBar = TRUE, # the change I asked you to make
importer = read_tsv
)
Yes. I tested it again,
> Txi_gene <- tximport(
+ path,
+ type = "none",
+ tx2gene = clean_tx_gencode_gtf,
+ txOut = FALSE,
+ countsFromAbundance = "lengthScaledTPM",
+ txIdCol = "target_id", # Specify the correct column name for transcript IDs
+ abundanceCol = "tpm",
+ countsCol = "est_count",
+ lengthCol = "transcript_length",
+ ignoreTxVersion = TRUE,
+ ignoreAfterBar = TRUE,
+ importer = read_tsv
+ )
Rows: 252913 Columns: 5
Column specification
Delimiter: "\t"
chr (1): target_id
dbl (4): raw, est_count, tpm, transcript_length
Use `spec()` to retrieve the full column specification for this data.
Specify the column types or set `show_col_types = FALSE` to quiet this message.
Rows: 252913 Columns: 5
Column specification
Delimiter: "\t"
chr (1): target_id
dbl (4): raw, est_count, tpm, transcript_length
Use `spec()` to retrieve the full column specification for this data.
Specify the column types or set `show_col_types = FALSE` to quiet this message.
Error in tximport(path, type = "none", tx2gene = clean_tx_gencode_gtf, :
all(txId == raw[[txIdCol]]) is not TRUE
Also, you did not follow Josie's recommendations fully, as you changed the value for the txIdCol parameter but not the corresponding colname of the tsv data. Nor did you include just the transcript_id and est_count columns like they asked you to, so you have not followed anyone's suggestions completely. Doing just a part of what people ask you to do will just give you different errors and annoy people trying to help you.
Apologies for bothering you. I appreciate your help for fixing this issue. Josie asked to use only two columns 1. target_idand2. est_count columns from NanoCount. I did exactly as she suggested but it did not help. Instead of removing those columns from the input tsv files, I just commented out those option out to ignore them. Please correct me if I am wrong. It was my mistake to leave transcript_id in tximport command but I changed it to target_id. Still it is showing error.
Log in to answer this question.
-
tximport error (medianLengthOverIsoform)
written by LGG •
-
Extract transcript ID and gene ID from ITAG4.1_gene_models.gff
written by Tian •
-
feature count command error
written by Adyasha •
-
bioawk command fails to generate tx2gene.tsv
written by Claire •
-
Error with Rseqc infer_experiment - unknown datatype
written by newbie
-
error in TXIMPORT command for RSEM
written by StartR •
-
Counting how many transcripts per gene in gtf file
written by sallyey2 •
-
How to create tx2gene object with gene names from annotation
written by poecile.pal •
-
Unable to convert txt to gtf format
written by Hyper_Odin
-
GTF2BED does not provide meaningful output?
written by c_u
What was the exact error? This should have worked.
Is it possible to know the solution of above discussed issue. Since I am also experiencing the error as below:
Thanks.