I"m a new to Galaxy. I’m trying to analyze my RNA seq data from mice using HISAT2 (or STAR) and featureCount. My data are stranded paired end. I could get a good mapping rate in HISAT2, but only 30% of reads were assigned in featureCounts. HISAT2 (with default settings except for strandedness, R…

It could be either one, you’ll need to do some more detective work. Is there someone at the sequencing facility you can talk to? Examples: gDNA contamination could be in the original sample (and would presumably be checked for by whoever is doing the library prep). rRNA contamination could be intro…

Hi, [image] Satsea: HISAT2 (with default settings except for strandedness, R) The strand for HISAT2 paired-end inputs should be FR, RF, or Unstranded, so this might be a typo and you meant RF? Featurecounts also requires strandedness to match what was used for mapping. That is an F, R, or U…

I really appreciate your help. The strand for HISAT2 paired-end inputs should be FR , RF , or Unstranded , so this might be a typo and you meant RF ? Yes, it is RF. Sorry for the typo. I selected RF for HISAT2 and R for featureCounts. I judged the strandedness by Infer Experiment. [25] D…

[image] Satsea: I selected RF for HISAT2 and R for featureCounts. I judged the strandedness by Infer Experiment. This looks correct if the bed dataset used with Infer was from the correct genome/build. You might want to double check that. This tutorial covers more details about strandedness…

Thanks a lot. I have organized my history in Galaxy Main. Now it may be a little bit easier to see. I couldn’t send you mail although I tired. Could you find my history using my e-mail address? I’m using the same address both in Galaxy Help and Main. I double checked the result of Infer using a …

Hi, Ensembl chromosome identifiers are different than those in the UCSC mm10 genome. The Gencode and iGenomes versions will work. FAQ: Mismatched Chromosome identifiers (and how to avoid them) Did you visualize the HISAT2 bam and reference annotation (UCSC’s bed, plus whatever gft you decide to us…

I see. I will run STAR again with the proper identifiers. I hope it works. Thank you. As for HISAT2 > feature counts, I only used the built-in data. If you can find out something wrong as to the HISAT2>featureCounts process in the history, please tell me how to solve it.

Hi, I ran STAR with a GFF3 file from Gencode. It worked, but the results by featureCounts using the data was similar to those by HISAT2. HTSeq generated the lots of no_features as well. STAR>featureCounts [52] STAR> HTSeq [06] Now, I’m trying to visualize the bam file by IGV. I have followed…

Hi - I took a look at your history. The problem is with the sequence content. Examine the FastQC results and you’ll see a very large number of sequences in the “Overrepresented” and “Sequence Duplication” reports, even for the version of the data that had adaptor removed. Some of these are a single…

Thank you very much indeed. Now, I’m relieved a little bit. This library was built by a specialized facility in our university. Do you think the problem is due to the quality of samples we submitted or the way they constructed the library?

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How can I improve very low assigned rate in featureCounts?

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