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  • FASTQ workflow on Miseq

    Hello dear colleagues,
    I have run an analysis on a Miseq system and it ended successfully.
    Before the run, I have set up "generate FASTQ only" as my workflow, because I plan to use another software to align my data to a reference genome.
    After the sequencing terminated, I checked my data and find out that FASTQ were not generated.
    My folders look exactly like the secondary analysis was not done. So, i opened Miseq reporter to try to requeue my analysis and complete it but it was not even listed there.
    In similar situations usually the error come from missing of "CompletedJobInfo.xml" and/or RTAComplete.txt
    In my running folder, I found "RTAComplete.txt" and "CompletedJobInfo.xml" was missing.
    I tried to copy and paste the "CompletedJobInfo.xml" from another run and reflesh the Miseq reporter, but still, it was not listed in completed run.
    So, I wonder if anyone of you has undergone a similar situation and if yes how he/she managed to resolve it.
    Thanks for any reply & help

  • #2
    Originally posted by Sergioo View Post
    After the sequencing terminated, I checked my data and find out that FASTQ were not generated.
    Is the "termination" above referring to normal run completion or was there an error of some kind?

    My folders look exactly like the secondary analysis was not done. So, i opened Miseq reporter to try to requeue my analysis and complete it but it was not even listed there.
    Have you looked in the D:\Illumina\MiSeqout or D:\Illumina\MiSeqtemp folders to see if the flowcell folder is there.
    In similar situations usually the error come from missing of "CompletedJobInfo.xml" and/or RTAComplete.txt
    In my running folder, I found "RTAComplete.txt" and "CompletedJobInfo.xml" was missing.
    I tried to copy and paste the "CompletedJobInfo.xml" from another run and reflesh the Miseq reporter, but still, it was not listed in completed run.
    So, I wonder if anyone of you has undergone a similar situation and if yes how he/she managed to resolve it.
    Thanks for any reply & help
    Those two files get written after successful completion of the basecalling/demultiplexing process. If those files are not present then that indicates that either RTA or some other process is not completing properly.

    Have you tried to contact Illumina tech support? At this stage they would probably be your quickest bet since they can remote in to your MiSeq and have a look at the run/analysis.

    Comment


    • #3
      The "termination" mentioned was a normal run completion and no error was reported.
      I contacted illumina tech support and will update when this issue is resolved.
      By the time being I think that it is related to the "generate FASTQ only" workflow of Miseq that is not working properly.
      Thanks

      Comment


      • #4
        The FASTQ files will be in

        D:\Illumina\MiSeqAnalysis\<RunFolderName>\Data\Intensitites\Basecalls\

        Comment


        • #5
          Originally posted by kmcarr View Post
          The FASTQ files will be in

          D:\Illumina\MiSeqAnalysis\<RunFolderName>\Data\Intensitites\Basecalls\
          FASTQ files was missing and CompletedJobInfo.xml as well.
          By searching deep into the folders, I found that the sample sheet was not recognized by the Miseq control software (for unknown reason, it was present in the Miseq control software without being copied into the analysis folder).
          I then decided to create another sheet (sample sheet.csv) with "assembly" as workflow, and I have copy-pasted that sample sheet into my Miseq analysis running folder and immediately the Miseq reporter recognized my run, which allowed me to to re-queue the run from there and it was completed normally.
          As you understand (as much as you can given that my English is broken, sorry), the issue was not solved and I stick on my assumption that there might be a problem with "generate FASTQ only" workflow.
          I would really be happy to listen from people who did it before and successfully.

          Comment


          • #6
            I've never had any problems with the "generate FASTQ only" workflow. How did you create your sample sheet? Do you still have it?

            Comment


            • #7
              Originally posted by dfhdfh View Post
              I've never had any problems with the "generate FASTQ only" workflow. How did you create your sample sheet? Do you still have it?
              I have created the sample sheet using the IEM as it is usually done.
              Create sample sheet------Other-----Generate FASTQ only-----select the plate------and check whether everything was ok, then finished.

              I don't think the error was from the sample sheet per se. I have sent it to illumina no problem was found

              Comment


              • #8
                If the "assembly" workflow worked with the new sheet then it may not hurt to try the "generate FASTQ" one again.

                Comment


                • #9
                  Originally posted by GenoMax View Post
                  If the "assembly" workflow worked with the new sheet then it may not hurt to try the "generate FASTQ" one again.
                  Dear Colleagues,
                  Thank you for your contributions!
                  I want just to close to update you on what happened.
                  I got assistance from Illumina and they just created another sheet for me.
                  In fact, they could not detect what was wrong with mine and they simply proposed me a new one.
                  When I copied the illumina-made sheet, immediately the run reporter recognized the run and went on to finish generate FASTQ files as intended for my experiments.
                  So, everything finished well, except tha I do not know if I will be asking illumina to provide me with a sheet whenever i run Miseq 7
                  Thanks all.
                  regards,

                  Comment


                  • #10
                    I'm a bit late to this thread, but we had the same problem with the FASTQ workflow (the only one we run). We found that the failure to use the sample sheet for the analysis was due to using a period/decimal in the 'Reagent Cartridge Barcode' field.

                    Despite the fact that IEM allows you to use a '.' in that field (which eventually becomes the name of the sample sheet if you use the default name suggested by the IEM software), having a '.' in the sample sheet name reproducibly causes FASTQ generation to fail.

                    Comment


                    • #11
                      Originally posted by RedSycamore View Post
                      I'm a bit late to this thread, but we had the same problem with the FASTQ workflow (the only one we run). We found that the failure to use the sample sheet for the analysis was due to using a period/decimal in the 'Reagent Cartridge Barcode' field.

                      Despite the fact that IEM allows you to use a '.' in that field (which eventually becomes the name of the sample sheet if you use the default name suggested by the IEM software), having a '.' in the sample sheet name reproducibly causes FASTQ generation to fail.
                      You are right. Thanks for your contribution

                      Comment


                      • #12
                        Just today, I had an issue with the fastq files generation. At the end of sequencing, MiSeq didn't generate these files, so I run MiSeq Reporter for that task, but it didn't work. Then I discovered the "RTAComplete" file was not in the folder it should be (neither Analysis nor Output folder), and in my BaseSpace... the run was not really finished! It appeared as "Analyzing", though in MiSeq it had already ended yesterday. It was weird, but after a while, finally the run showed at BaseSpace as "Complete", and I could generate all fastq files.
                        I hope my experience could be of help for someone with the same problem
                        Science is ok, but I'm hungry.

                        Comment


                        • #13
                          BaseSpace can take a reeaalllly long time to analyze. I talked to Illumina about it a few months ago after it had taken more than 7 hours to generate FastQ files. Their response was that sometimes BaseSpace doesn't start the analysis until several hours after the run finishes. It says analyzing, but in reality it's in queue to be analyzed.

                          Comment


                          • #14
                            Also ArciMol is from Chile, so I am not sure what kind of bandwidth (s)he has available to get the data to BaseSpace.

                            On a different note: I understand that a TB of space is offered for free on BaseSpace. What is the attraction of using BaseSpace (as opposed to the on-board analysis), specially for generation of just fastq files? Are people actually using paid apps available on BaseSpace (which can be one real reason to use it).

                            Comment


                            • #15
                              We're a small core so BaseSpace is an easy way to distribute data. It's also nice for me to be able to keep track of how the run looks in real time from home. None of our investigators actually use BaseSpace to analyze their data.

                              Comment

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