Moderator: Charles Warden
Charles Warden ♦ 5.2k
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I am a Bioinformatics Specialist at City of Hope, where I work in the Integrative Genomics Core doing microarray and high-throughput sequencing analysis.
Posts by Charles Warden
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... I think [rnaseqGene][1] has a very nice DESeq2-centric tutorial with a helpful [visualization][2] and [explanation][3].
At least for me, this difference wasn't as clear from reading the papers, but the VST normalized counts in that figure look more like thresholded counts.
[1]: http://master.bi ...
written 15 hours ago by
Charles Warden ♦ 5.2k
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... [FastQC][1] will give over-represented sequences with some Illumina library sequences (if you ended sequencing a bunch of adapter sequences, for example), and it can also include polyA/polyT sequences. It will also give some overall GC sequence content information.
[FastQ Screen][2] can also help ...
written 19 hours ago by
Charles Warden ♦ 5.2k
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... If you are using GENCODE annotations, it should be OK to use both coding and non-coding gene annotations (unless you were doing something like using RefSeq coding genes and GENCODE lncRNAs).
My experience has been in a slightly different context, so I don't know if it relevant for your project. Ho ...
written 19 hours ago by
Charles Warden ♦ 5.2k
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... To be honest, I've typically parsed the counts from [Salmon][1] / [Sailfish][2] directly (when I tried RSEM, the alignment either took a long time, or the quantification didn't seem as good: however, I assume that you have had better luck...).
However, if you are worried about bugs in your custom c ...
written 1 day ago by
Charles Warden ♦ 5.2k
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... I haven't personally tested [ANCOM][1], but I've tested [MetagenomeSeq][2].
I think it would be safe to use an overlap between methods, but you may find that too conservative in some cases.
If you have some way to justify a particular choice (to match your overall feel for results, or validate a k ...
written 1 day ago by
Charles Warden ♦ 5.2k
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... If you use different annotation sources, separate quantification can avoid issues with ambiguous reads in overlapping annotations from the different sources (where a similar or related overlapping annotation has a different name in the different annotation files). Ideally, having a merged list prio ...
written 1 day ago by
Charles Warden ♦ 5.2k
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... I would typically filter out cells with a low coverage of genes (either without any reads, or genes with CPM below a certain threshold), as well as some total number of reads per cell.
If you only look at cells with say >50% gene coverage, I don't think imputation would be as important (presumab ...
written 1 day ago by
Charles Warden ♦ 5.2k
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... If it is a polyA enriched protocol, you might have had low RNA input (or an extra high number of target sequences) if you see mostly polyT/polyA reads.
There may be other GC-centric metrics that other people find useful. If the genome was small and contamination was high, you might be able to BLAS ...
written 1 day ago by
Charles Warden ♦ 5.2k
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... Thank you for sharing that information.
In that plot, it looks like the linear model (which I believe you expect to comparable to the `glm()` generalized linear model in this discussion) is similar (if not slightly better) than the more complicated model.
In general, I think this is interesting be ...
written 1 day ago by
Charles Warden ♦ 5.2k
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Comment:
C: About aligment in RNASeq analysis
... While I've heard this opinion a lot, I don't think this is necessarily true:
1) I know of at least one project where I needed to use a TopHat2 alignment to recover a known splicing event. So, there may be rare cases where it is helpful to use TopHat, particularly if the downstream program is devel ...
written 2 days ago by
Charles Warden ♦ 5.2k
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