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Results tagged with Search options user 29

Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...

3
votes
Neither CPM nor TPM are well suited here, because neither performs robust cross-sample normalisation (see the blog post Devon linked to). DESeq2 provides two robust log-space normalisation methods fo …
answered Aug 15 '17 by Konrad Rudolph
10
votes
2answers
I’d normally use collapseReplicates (or do the collapsing upstream) to handle technical replicates. However, in my current RNA-seq experimental design, samples were sequenced twice using different li …
asked May 24 '17 by Konrad Rudolph
11
votes
5answers
I’ve got an RNA-seq dataset with a large proportion of environmental RNA “contamination”. BLASTing random reads reveals that much of the data comes from bacterial, plant and viral RNA. My target organ …
asked Aug 1 '17 by Konrad Rudolph
10
votes
I'd like to find genes that were not expressed in a group of samples and were expressed in another group. This is, fundamentally, a differential expression analysis, with a twist. To solve this, …
answered Jun 13 '17 by Konrad Rudolph
4
votes
I have seen many posts regarding counts to RPKM and TPM. There’s your answer then: FPKM = RPKM. It’s simply a more accurate name. Speaking of RPKM for paired-end data is discouraged because the …
answered Oct 22 '18 by Konrad Rudolph
4
votes
Can’t be done. If you already sequenced then I’m afraid the money is wasted (unless of course the data is good for something else). The standard Illumina basecaller doesn’t deal well with homopolymer …
answered Dec 1 '17 by Konrad Rudolph
30
votes
First off, Don’t use RPKMs. They are truly deprecated because they’re confusing once it comes to paired-end reads. If anything, use FPKMs, which are mathematically the same but use a more correct na …
answered May 17 '17 by Konrad Rudolph
2
votes
When sequencing RNA from freshly harvested tissues under proper conditions, you should generally expect > 50% mapped reads. In fact, everything < 80% would usually raise concerns. From your descripti …
answered Nov 23 '17 by Konrad Rudolph
4
votes
Try reducing the number of threads. When multithreading the index creation (and other memory bound tasks), the memory usage increases linearly with the number of threads. If you required a 10 GiB wor …
answered May 24 '18 by Konrad Rudolph
3
votes
You should never use RPKM. It’s simply obsolete in the age of paired-end sequencing, and has been replaced by FPKM (which is, strictly speaking, a synonym). The linked blog post explains more general …
answered May 9 '18 by Konrad Rudolph