QC Error

[hector3500]

When I try to conduct the QC on my experiments the BAM files output fine, get converted to ofst files, and have read length tables created for them, though an error keeps popping up after this step:

ORFikQC(df.rfp) Started ORFik QC report for experiment: Riboseq_1_Drosophila Saving, ofst files to: /home/harrison/Documents/RiboSeq/Untrimmed_reads/Trimgalore_reads/PolA_trimmed/Mapped/Sorted/Unique_2/ofst/ Outputting libraries from: Riboseq_1_Drosophila

Converting libraries to new format: ofst RFP_RPS3_r1 fstcore package v0.9.14 (OpenMP detected, using 8 threads) RFP_RPS3_r2 RFP_RPS3_r3 RFP_RPS3.XRP1_r1 RFP_RPS3.XRP1_r2 RFP_RPS3.XRP1_r3 RFP_WT_r1 RFP_WT_r2 RFP_WT_r3

• Creating read length tables: Outputting libraries from: Riboseq_1_Drosophila Outputting libraries from: Riboseq_1_Drosophila Error: BiocParallel errors 5 remote errors, element index: 1, 3, 5, 7, 9 4 unevaluated and other errors first remote error: Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'seqlevelsStyle': cannot open the connection

[Roleren]

Hey, the error: "cannot open the connection", means file paths are wrong most likely.

Can you send me the information of file paths you defined in your ORFik experiment, that is

filepath(df, "default") # <- df is the ORFik experiment

Also, could you try this:

rfp <- fimport(filepath(df, "default")[1]) # Load first bam file directly
seqlevelsStyle(rfp) # Check seqlevels Style can be read correctly

[hector3500]

Hey, I'll just give you the full experiment:

create.experiment( file.path("/home/harrison/Documents/RiboSeq/Untrimmed_reads/Trimgalore_reads/PolA_trimmed/Mapped/Sorted/Unique_2"), exper = "Riboseq_1_Drosophila", fa = "genome", txdb = "gtf", organism = 'Drosophila melanogaster',
viewTemplate = FALSE, pairedEndBam = FALSE, rep = c(1,2,3,1,2,3,1,2,3), condition = rep(c("RPS3", "RPS3.XRP1","WT"), each = 3), libtype = "auto", stage = "auto" )

I already defined the genome and gtf paths previously.

[Roleren]

Yeah, so that wont do, I need to see the actual output, and this must also be wrong, as this will not work: txdb = "gtf"

it should this I guess?: txdb = gtf

[Roleren]

I think I need the specified outputs I required above to be able to know what is going on, as your definition above hides too many details

[hector3500]

okay so the genome and gtf paths are: genome = "/home/harrison/Documents/RiboSeq/Genome/Drosophila_melanogaster.BDGP6.46.dna_sm.toplevel.fa" gtf = "/home/harrison/Documents/RiboSeq/Genome/Drosophila_melanogaster.BDGP6.46.57.gtf"

I removed the quotation marks, still the same error though. When you say outputs do you mean the paths for the output ofst and readLengths file? I didn't define output paths, it automatically places them in the file.path directory I initially specified.

[hector3500]

Here's the rest of the commands follwing the created experiments (I also have an RNAseq experiment):

df.rfp <- read.experiment("Riboseq_1_Drosophila") df.rna <- read.experiment("RNAseq_1_Drosophila")

ORFikQC(df.rfp) ORFikQC(df.rna)

[Roleren]

Yes, and show me the console output when you run these lines:

df.rfp <- read.experiment("Riboseq_1_Drosophila")
filepath(df.rfp, "default") # <- df.rfp is the ORFik experiment
rfp <- fimport(filepath(df.rfp, "default")[1]) # Load first bam file directly
seqlevelsStyle(rfp) # Check seqlevels Style can be read correctly

[Roleren]

Any more questions? Else I will close the issue :)