Question: Rna-Sea analysis and cufflinks
0
gravatar for ms5168
22 months ago by
ms51680
ms51680 wrote:

Hello,

I am using the following pipeline to analyze my RNA-Seq data to detect differences in gene expression:

FASTQ Groomer -> TopHat -> Cufflinks -> Cuffmerge -> Cuffdiff -> CummeRbund

Do I miss anything in the pipeline or this is the correct workflow for gene expression analysis?

Also,this might be a trivial question, but when using cuffmerge it supposed to merge the two files (assembled transcripts) produced by cufflinks. However, when I try to click on two files, only one of them is marked as blue. When I try to add "Insert Addition GTF Input", nothing comes up on the list. In other words, how do I pick the two assembled transcripts files to be merged by cuffmerge?

Thanks,

Moti

rna-seq cuffmerge cufflinks • 577 views
ADD COMMENTlink modified 22 months ago • written 22 months ago by ms51680
0
gravatar for Jennifer Hillman Jackson
22 months ago by
United States
Jennifer Hillman Jackson24k wrote:

Hello,

Use "command-click" to select multiple datasets in the first data input box. The second is for a different datatype (collection lists).

Let us know if this does not work, Jen, Galaxy team

ADD COMMENTlink written 22 months ago by Jennifer Hillman Jackson24k
0
gravatar for ms5168
22 months ago by
ms51680
ms51680 wrote:

It works greatly, thank you.

One more question, how can I tell if my sequence data is single-end or paired-end?

Thank you very much

ADD COMMENTlink written 22 months ago by ms51680

How many input fastq datasets to did you have for each sample/replicate? Paired end data in fastq format will be in pairs of datasets (BAMs may be merged). For either, the sequence IDs will share an identifier designating which fastq is the forward or reverse read of the pair.

Also see: https://wiki.galaxyproject.org/Learn/GalaxyNGS101

Plus other help at the top of the Galaxy Support wiki: https://wiki.galaxyproject.org/Support

ADD REPLYlink written 22 months ago by Jennifer Hillman Jackson24k
0
gravatar for ms5168
22 months ago by
ms51680
ms51680 wrote:

Thank you for the prompt reply, Jennifer.

I recently started to work in this lab. All the RNA preps were created and sent for sequencing prior to my arrival. Some directories have one fastq file while others have 2. Can I assume that the directories with 2 files are pair-end and the ones with 1 file are single-end?

If I view the file, what would tell me if it is a single/pair-end?

Thank you, this is very helpful.

Moti

ADD COMMENTlink written 22 months ago by ms51680

The link I sent explains how fastq identifiers can designate forward and reverse reads. But this won't answer the question "I found one file, how can I be certain there isn't a second?". I would contact your lab members that generated the data. Methods should be captured and shared with you somewhere - or resulting analysis will not be suitable for extracting meaningful conclusions from the results (including publication).

ADD REPLYlink written 22 months ago by Jennifer Hillman Jackson24k
0
gravatar for ms5168
22 months ago by
ms51680
ms51680 wrote:

Thank you very much, Jennifer.

This is very helpful.

I have another issue.

I ran the following pipeline: Alignment -> Cufflinks -> Cuffmerge -> Cuffdiff -> CummeRbund (failed)

After running Cufflinks, I checked to see that there are FPKM values in the "transcript.gtf" files and that the values are not 0 (they were not). However, after merging the files and running cuffdiff, the "gene differential expression testing" file showed 0 for value_1 and value_2 and "NOTEST" for the status. This is true for the entire file.

Any idea what went wrong?

Thank you.

Moti

ADD COMMENTlink written 22 months ago by ms51680
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