Help With Sleuth
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8 days ago
Rangarajan • 0

Hello,

I apologize if this is a simple post. I am trying to study the differential expression of Borrelia Burgdorferi on Sleuth to practice skills. However, I am unable to find transcripts on Enseml using the tutorial Lior Pachter posted. Would someone be able to guide me to how I can get annotated transcripts and edit my sleuth object?

I have been using https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000008685.2/ thus far.

Thank you.

Sleuth RNASeq • 244 views
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This isn't nearly enough information to help you. What do your current identifiers in your object look like and what does your annotation file look like? What tutorial are you referring to? If it's a tutorial by Lior, probably be best served by asking his group directly via whatever support mechanisms they have for their tooling.

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My annotation file has been the https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/008/685/GCF_000008685.2_ASM868v2/GCF_000008685.2_ASM868v2_genomic.gtf.gz

It seems to be the right one? I indexed it based off of the cds file from the same genbank link

The tutorial is

https://pachterlab.github.io/sleuth_walkthroughs/trapnell/analysis.html

For my analysis I am looking at four pleomorphic variants of borrelia and seeing their transcriptional variation. The paper used Deseq but it appears sleuth is better.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10057712/

In making my sleuth object I got,

normalizing est_counts 1337 targets passed the filter normalizing tpm merging in metadata summarizing bootstraps

the models are [ full ] formula: ~condition data modeled: obs_counts transform sync'ed: TRUE coefficients: (Intercept) conditionbleb conditionround_body conditionspirochete [ reduced ] formula: ~1 data modeled: obs_counts transform sync'ed: TRUE coefficients: (Intercept)

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I would actually recommend using edgeR's catchKallisto option over sleuth based on recent benchmarks from this year. (I work with Pachter FYI).

Nonetheless, sleuth still gives good results, and I don't see any issue you're having. You've made your sleuth object via sleuth_prep, you've run sleuth_fit to run your models, now just go ahead and run sleuth_results. You already have your transcripts; it's what in your index when you mapped your reads.

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