Alhamdulillah. I have followed all of steps guided by you and successful in my first attempt. Take my heartiest love 💕 for your assist. Half of my project is done by the help of the video.
That was very insightful. I am going to be doing an MSc in bioinformatics and biotechnology in September, so from a beginner's point of view, you explained everything in detail which was very helpful. Thank you.
Thank you so much for this tutorial. It helped me understand the steps to paired end data analysis. I have a doubt, on running trimmomatic 6:48 we get paired and unpaired file for all entries (R1 and R2 of treated and control), later on we do FASTQC for both paired and unpaired trimmomatic outputs (8:25) and we takeup paired trimmomatic files for HISAT2. As you said we do not really need it. So, I guess we can too skip doing FASTQC with the unpaired files?
Thank you so much for this tutorial! I am doing the steps for bacterial RNA seq data analysis and will thus be using my own reference genome and GTF file for HISAT2 and featurecounts. However, that does not seem possible for the tool annotatemyIDs, since there isnt an option for me to upload an annotation file. What can I do in this case to proceed with Limma?
Hello Prof. Fathima, Many thanks for this valuable and versatile vidoe. After trimming, if there is an error and the output file turns red, what should be done? In your File 104, what does "A list with 1 item" mean? In my own case, it shows "A list with 0 datasets."
hello madam..i m facing an issue. I cannot find my organism "PLANT" after the annotatemyIDs step and when i give ensembl gene option it shows error...How to rectify this issue..I uploaded plant genome from history....need your help...thanks
Thank you for the video, you helped a lot. I have just one question, should we select "Perform initial ILLUMINACLIP step" on trimmomatic tool to cut adapter sequences? Cuz my raw data includes adapters as well.
Hi Dr. Fathima: In addition, is it possible to run one control and two treated files for gene expression pattern with Limma? I did so and it resulted in an error.
that was useful,but why I had no result and error when I conduct LIMMA, An error occurred with this dataset: [1] "Extracting counts" [1] "Generating DGEList object" [1] "Generating Design" [1] "Calculating Normalisation Factors" [1] "Extracting counts" [1] "Generating DGEList object" [1] "Generating Design" [1] "Calculating Normalisation Factors" Error in if ( I appreciate a lot if you could help me.
¿Como añado un archivo gff o gtf ?lo he intentado pero algo sale mal y los conteos me dan cero, he confirmado que el genoma es el correcto pero los parámetros no se si son correctos?
Hello Daniel, I am not very familiar in how to use gff files with featurecounts but try converting the gff files to BED files and then to BAM files. I believe there are workflows on Galaxy that do this, just search "GFF to BED" and then "BED to BAM." Hope this helps!
@@fathimashaik1241 Hi Fathima, Thanks for the video. it worked for me the first time! However, annotatemyids did not work due to the fact that the Galaxy do not have my species (Arabidopsis) gene annotation file. I have uploaded a few files, but none worked. I tried converted gff files to BED, but could not convert BED to BAM. Galaxy do not have BED to BAM converter. Closest one I found is bedtools BEDPE to BAM and it did not work. Can you help? Thanks
This was so helpful! thank you! Can you please show how you create a cluster map from this? I've tried running scanpy plot t-sne and then using it for input into scanpyplot embed but its failing :(
At 29:17 you can see at the top of column D is the gene name and instead of deleting it, you can keep it. That way while you organize and sort the spreadsheet, the gene names will still be present when you determine which are upregulated and which are downregulated. Hope this helps!
@@fathimashaik1241 Thank you so much. could you please tell us why do you select the expressed gene score +2 and above and suppressed gene score -2 to less ?? I mean does it followed a standard rule??
HELP first of all thank you so much for this tutorial, im having a problem with featurecounts tool for a dataset for tomato. im encountering this error: failed to find the gene identifer in 9th column. i cut the empty space of the few first rows as was mentioned somewhere, but im still getting this error. can you please help me?
Hello, Are you using GFF files? If so, those files need to have a ref_gene_id column and yours might be missing this. To fix it, you can use the filter GFF by attribute and type "ref_gene_id". Then, upload the files again to the featurecounts and see if it fixes the issue. I assume you are using GFF files. If you are not then please reply back and I can suggest a new method. Hope this is helpful to you.
@@fathimashaik1241 thank you for answering. actually i used gff and gtf to see if one of them works. then i downloaded both gene reference file and annotation file from the same source but it did not help. then i deleted the comment (#) lines of my annotation file so my file begins with the gene_id column but it did not help either...
@@fathimashaik1241 hello, as you suggested I used the tool "Filter GFF by attribute" and in the "with following condition" I typed "ref_gene_id". it produced the following error; illegal/invalid in condition "ref_gene_id". am I doing it wrong?
annotateMyIDs does not support my organism (triticum aestivum). Is their any alternative for it. If I use Limma without the annotation file, it doesn't work because "plotMDS requires at least 3 columns" and my feature count files have 2 columns.
Hi! I don't know if you've figured this out yet but I have found the solution for that. I had this issue since my organism isn't on any of the predetermined lists. So I first, downloaded the genome from NCBI in .gtf format and input that into the "Gene annotation file" section of the featureCounts step. In my case, for the results of the featureCounts, counts results, it showed that my "Gene id" was actually the locus tag (any unique identifier should do, so on the right track) Using that knowledge, I looked for an annotation file (on NCBI) containing the locus tag as well as the important annotations. I downloaded that in a .tsv format (Excel) I opened up that file in Excel and made sure that it was formatted to a table, identifying the headers. Then I moved the "locus tag" or whatever unique identifier was observed in featureCounts, to the first column before any accession numbers, etc. (I tried just using the Excel file without this step and it does not work) Last, import the modified Excel file into the gene annotation file in the limma step, and it works! I would try refreshing about 3 times before you try something else! Good luck!
Sample fastq.gz files are found on different websites. You can just Google "sample fastq.gz files" and find hubs where free fastq.gz files are available to use and practice with. The data I used in this video were acquired from my lab's research.
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Alhamdulillah.
I have followed all of steps guided by you and successful in my first attempt.
Take my heartiest love 💕 for your assist.
Half of my project is done by the help of the video.
Glad I could help!
You must be the angel in this world. The video really helped me a lot.
Thank you, it was my pleasure to help you!
That was very insightful. I am going to be doing an MSc in bioinformatics and biotechnology in September, so from a beginner's point of view, you explained everything in detail which was very helpful. Thank you.
My pleasure! I'm so glad you found this video helpful.
Thank you. And please preview every output and demonstrate as you know up to detail. we expect more videos in future.
Absolutely insightful. Thank you very much for this
Thanks, this helped a bunch
Glad I could help! :)
Yes, I enjoyed it. Thank you so much, it was really good.
Glad you enjoyed it!
Thank you for making this great content
Hello Mam, thank you for sharing such a insightful video and your explanation was very detailed with explanation. It was great.
Really informative. Love from Pakistan. would you please continue to make vedioes like this, these would be really helpful. Always flourish 🥰🥰
Excellent! Thank you very much for your video!
You're welcome!
Thank you so much for this 💜
No problem!
Is it normal to have neg AveExpr levels at the same time as positive logC values?
Thank you so much for this tutorial. It helped me understand the steps to paired end data analysis. I have a doubt, on running trimmomatic 6:48 we get paired and unpaired file for all entries (R1 and R2 of treated and control), later on we do FASTQC for both paired and unpaired trimmomatic outputs (8:25) and we takeup paired trimmomatic files for HISAT2. As you said we do not really need it. So, I guess we can too skip doing FASTQC with the unpaired files?
Thank you so much for this tutorial! I am doing the steps for bacterial RNA seq data analysis and will thus be using my own reference genome and GTF file for HISAT2 and featurecounts. However, that does not seem possible for the tool annotatemyIDs, since there isnt an option for me to upload an annotation file. What can I do in this case to proceed with Limma?
Please please pleasee post more videos for post processing to show how we can do all the visualizations (venn diagram, volcano plots etc.)
Really useful video
Thank you! Glad I could help.
Excellent Tutorial !!! Please uplaod same analysis using LINUX environment
Excellent 👍🏼
Thank you!
great great great 👌👌👌
Hello Prof. Fathima, Many thanks for this valuable and versatile vidoe. After trimming, if there is an error and the output file turns red, what should be done? In your File 104, what does "A list with 1 item" mean? In my own case, it shows "A list with 0 datasets."
hello madam..i m facing an issue. I cannot find my organism "PLANT" after the annotatemyIDs step and when i give ensembl gene option it shows error...How to rectify this issue..I uploaded plant genome from history....need your help...thanks
Hello, unfortunately, I don't believe that Galaxy has the option to choose the organism "Plant". This may be why the error occurred.
Great tutorial!
Hi Dr. Fathima, Also, how can I change the FDR from 0.05 to 0.1? Thank you.
thanks, very nice. Is there a GEO posting for your data? thanks.
Unfortunately I do not have one, sorry about that.
Hi! Can we have a link for the Fasta files you used in this video? Thanks!
Thank you for the video, you helped a lot. I have just one question, should we select "Perform initial ILLUMINACLIP step" on trimmomatic tool to cut adapter sequences? Cuz my raw data includes adapters as well.
hi, many thanks for your professional video.May I ask you give me link for your data, contral and treated samples, fastq files?
Hi Dr. Fathima: In addition, is it possible to run one control and two treated files for gene expression pattern with Limma? I did so and it resulted in an error.
Can you please help? Why am I not able to perform limma step? Evertime i execute it, it comes back with an error.
Sure, could you please elaborate on your issue? What steps did you execute beforehand?
that was useful,but why I had no result and error when I conduct LIMMA, An error occurred with this dataset:
[1] "Extracting counts"
[1] "Generating DGEList object"
[1] "Generating Design"
[1] "Calculating Normalisation Factors"
[1] "Extracting counts"
[1] "Generating DGEList object"
[1] "Generating Design"
[1] "Calculating Normalisation Factors"
Error in if (
I appreciate a lot if you could help me.
Unfortunately, I have same error also with Limma!
Hello,
I think that the problem may be due to a faulty annotation file. Are you using the pipeline featureCounts and then annotateMyIDs?
Can you suggest me a pipeline to analyze preprocessed scRNAseq data using Galaxy?
¿Como añado un archivo gff o gtf ?lo he intentado pero algo sale mal y los conteos me dan cero, he confirmado que el genoma es el correcto pero los parámetros no se si son correctos?
Hello Daniel, I am not very familiar in how to use gff files with featurecounts but try converting the gff files to BED files and then to BAM files. I believe there are workflows on Galaxy that do this, just search "GFF to BED" and then "BED to BAM." Hope this helps!
@@fathimashaik1241 Hi Fathima, Thanks for the video. it worked for me the first time! However, annotatemyids did not work due to the fact that the Galaxy do not have my species (Arabidopsis) gene annotation file. I have uploaded a few files, but none worked. I tried converted gff files to BED, but could not convert BED to BAM. Galaxy do not have BED to BAM converter. Closest one I found is bedtools BEDPE to BAM and it did not work. Can you help? Thanks
This was so helpful! thank you! Can you please show how you create a cluster map from this? I've tried running scanpy plot t-sne and then using it for input into scanpyplot embed but its failing :(
Thank you so much. I have a question: how coulD I FIND the gene name that are upregulated or down regulated??
At 29:17 you can see at the top of column D is the gene name and instead of deleting it, you can keep it. That way while you organize and sort the spreadsheet, the gene names will still be present when you determine which are upregulated and which are downregulated. Hope this helps!
@@fathimashaik1241 Thank you so much. could you please tell us why do you select the expressed gene score +2 and above and suppressed gene score -2 to less ?? I mean does it followed a standard rule??
HELP
first of all thank you so much for this tutorial,
im having a problem with featurecounts tool for a dataset for tomato. im encountering this error:
failed to find the gene identifer in 9th column.
i cut the empty space of the few first rows as was mentioned somewhere, but im still getting this error.
can you please help me?
Hello,
Are you using GFF files?
If so, those files need to have a ref_gene_id column and yours might be missing this. To fix it, you can use the filter GFF by attribute and type "ref_gene_id". Then, upload the files again to the featurecounts and see if it fixes the issue. I assume you are using GFF files. If you are not then please reply back and I can suggest a new method. Hope this is helpful to you.
@@fathimashaik1241 thank you for answering. actually i used gff and gtf to see if one of them works. then i downloaded both gene reference file and annotation file from the same source but it did not help. then i deleted the comment (#) lines of my annotation file so my file begins with the gene_id column but it did not help either...
@@hhhh56hh545 Hello, did the above method for GFF files work for you?
@@fathimashaik1241 hello, as you suggested I used the tool "Filter GFF by attribute" and in the "with following condition" I typed "ref_gene_id".
it produced the following error; illegal/invalid in condition "ref_gene_id".
am I doing it wrong?
fathima when everthing is denovo how to generate gff or gft file for featurecount
AnnotateMYiD is not working for human genome
''An error occurred with this dataset:
format tabular database hg38''
may you kindly help me with this?
Having trouble with the limma tool. Its showing zero data sets.
please if anyone can help. needed urgently
annotateMyIDs does not support my organism (triticum aestivum). Is their any alternative for it. If I use Limma without the annotation file, it doesn't work because "plotMDS requires at least 3 columns" and my feature count files have 2 columns.
Hi! I don't know if you've figured this out yet but I have found the solution for that. I had this issue since my organism isn't on any of the predetermined lists.
So I first, downloaded the genome from NCBI in .gtf format and input that into the "Gene annotation file" section of the featureCounts step.
In my case, for the results of the featureCounts, counts results, it showed that my "Gene id" was actually the locus tag (any unique identifier should do, so on the right track)
Using that knowledge, I looked for an annotation file (on NCBI) containing the locus tag as well as the important annotations. I downloaded that in a .tsv format (Excel)
I opened up that file in Excel and made sure that it was formatted to a table, identifying the headers. Then I moved the "locus tag" or whatever unique identifier was observed in featureCounts, to the first column before any accession numbers, etc. (I tried just using the Excel file without this step and it does not work)
Last, import the modified Excel file into the gene annotation file in the limma step, and it works!
I would try refreshing about 3 times before you try something else!
Good luck!
Some of the audio is very low
Sorry about that! The audio encountered some issues when the video processed.
thanks for you user giude, can you help to me about meta analysis rnaseq data with combat in galaxy
from where we can download dataset
Sample fastq.gz files are found on different websites. You can just Google "sample fastq.gz files" and find hubs where free fastq.gz files are available to use and practice with. The data I used in this video were acquired from my lab's research.
hi m the audio is sort of not help full.
Sorry about that! The audio becomes much higher in the second half of the video.
@@fathimashaik1241 thanks, a lot this work is really helpfull for me as a begginer on the use of this tool.
@@rexferdinandtraifalgar3477 Great to hear!
This is very helpful. Thank you
this is fabulous!! thanks so very much.
Hello I have been having error in running the limma tool. The result is showing a list with zero data sets
please can you help