Help Assigning groups and comparisons for RNASEQ files

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beste...@gmail.com

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Mar 3, 2019, 10:38:05 AM3/3/19
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Dear Sir or Madam,


I hit error messages where no groups or files are found or if I do differently a similar error message.  
 
 In the first window of the AltAnalyze I call my experiment "RNASEQANA" I then designate the location of my four fastq files. Finally, I designate the output folder location.

As per instructions, into "Expression input" folder I created a file called "groups.RNASEQANA" i.e, "groups"  followed by the name of my experiment and still didn't work, I then did the same but added the individual fastq file names after "RNASEQANA" shown below and still didn't work.

 Is there some specific way to create the group files that I am doing incorrectly? Does it need to be an excel file? e.g., ending in .xls?  Am I using the correct expression input folder location to enter the groups? Finally, is there an easier way to assign groups to my fastq files? and also comparisons because it is likely when I get this to work finally it will error because no comparisons are assigned.

I attached a ppt file with screen shots of my workflow and the error messages. I tried several ways to get my groups assigned as you will see in powerpoint screen shots.
I consulted the instruction manual but there are issues with the images loading (http://www.altanalyze.org/help.htm) and instruction text did not help either. I uploaded four RNA seq FASTQ files (http://bigd.big.ac.cn/gsa/browse/CRA000215) into AltAnalyze. They are paired reads so actually only two samples.
I am running windows 10 home on 64 bit operating system
My fastq files names as follows:

1) "CRD038401_1" (read 1)
2) "CRD038404_1" (read 2)

3) "CRD038407_1" (read 2)
4) "CRD038415_1" (read 1)

The images on the wepbage are not loading on any browser.

Best, 

Brian Estevez
Children's Hospital of Philadelphia
AltAnalyze_report-20190302-190004.log
assigning RNASEQ files into groups.pptx

Nathan Salomonis

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Mar 3, 2019, 10:47:50 AM3/3/19
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Hi Brian,

Looks like you are almost there but there are a few things to clarify. First, from the screenshots, it looks like the program was first run on the fastq files, generated results folders and then reimported those Kallisto pseudoaligment results. Please note, additional details and instructions can be found in the AltAnalyze program DemoData folder under the Kallisto directory along with sample data.

There appear to be two potential issues:

1) it’s not clear if the fastq were correctly processed, given that the files in ExpressionInput are only 1kb in size. It is trying to re-import results (ExpressionInput/kallisto folder, where each sub folder should include the kallisto primary results), so the log file won’t reflect the how the Kallisto analysis did. If run successfully, genome coordinate BAM files along with bed files in the output directory should exist with information within them. You can delete the kallisto folder in ExpressionInput and it will re-analyze the fastq files from scratch.

2) the groups file you created is not in the ExpressionInput folder you screenshot. This may be the problem or the format. 

Can you send me your groups file and a screenshot of the folder containing the fastq files so I can make sure everything looks good? Please also send the summary file in the ExpressionInput file.

Best,
Nathan
<AltAnalyze_report-20190302-190004.log>
<assigning RNASEQ files into groups.pptx>
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