Question: Demultiplex file on MG-RAST server
0
gravatar for daniellegrimaldi
2.4 years ago by
UFRJ, Rio de Janeiro, Brazil
daniellegrimaldi0 wrote:

I'm trying to analyze 16S rRNA gene sequencing data (illumina miseq) using the MG-RAST server. I merged R1 and R2 files, but I can’t demultiplex it. I have a barcode file(ASCII) and the demultiplex option does not appear when this file is selected. When I select the R file (merged file), MG-RAST only lets me choose another file to do the "join" step. Does anyone know how to solve this problem? MG-RAST is able to separate after using the metadata file after submission of the work?

ADD COMMENTlink written 2.4 years ago by daniellegrimaldi0

Looks like you need to upload metadata file first followed by the two separate fastq files (unmerged). Then merge the files on the server.

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by genomax91k

I merged the sequences (R1 and R2) in MG-RAST. I uploaded metadata file. It seems correct until submission. In the last step of submission, the message "could not validate metadata" appears. I need to demultiplex sequences before, right? And for each barcode will a file be generated, is it correct? My metadata file has 36 samples. In the MG-RAST manual it is written "Barcode files will automatically show the button for demultiplexing", but for my barcode file (ASCII) this message does not appear. Actually, I have two problems, I can not demultiplex the sequences and the metadata can not be validated. I need help. I've been trying to do this for a month. I've watched several tutorials on youtube, but I'm stagnant in this task.

ADD REPLYlink written 2.4 years ago by daniellegrimaldi0

the message "could not validate metadata" appears

I would suggest looking into that. Did you try the tool suggested (metazen) to create the metadata file? This video tutorial seems to have necessary information.

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by genomax91k

I tried metazen. it didn't work. I've tried several different things. Mg-rast shows that the name of the submitted file is not compatible or could not be found ('Name of the sequence file submitted to MG-RAST'). If I put the same file name for 36 samples, a message appears notifying you that the file name cannot be repeated. But my 36 samples are in a single file. I cannot identify what I'm doing wrong. I mean, I have to do the demultiplex step, but it does not recognize my barcode file, although it is in the correct format (ASCII). And also, the only place where I can enter my forward and reverse primers information is in metadata file. And it seems to me that this column "Name of the sequence file submitted to MG-RAST" is not fundamental either (it is not red in the worksheet). I'm totally lost!

ADD REPLYlink written 2.4 years ago by daniellegrimaldi0

When I select R file (with merged sequences), it gives me the options "demultiplex" or "join paired ends", I select "demultiplex" and the server asks me to select another file to "join paired ends".

ADD REPLYlink written 2.4 years ago by daniellegrimaldi0
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