running run-dropest

[bxxu]

Hi, I am having trouble trying to follow the tutorial (http://velocyto.org/velocyto.py/tutorial/cli.html#run-dropest-run-on-dropseq-indrops-and-other-techniques). The data I was working with were prepared using celseq2 with bp1-bp6 as UMI and bp7-bp12 as cell barcodes. I was able to follow the first part using dropest, obtaining count matricies (.rds) and a sortedByCoord.bam file from dropest.

However, problem arises when I run velocyto tools dropest-bc-correct. It appears that one potential causes of the problem came from the fact that rpy.rinterface does not contain the RNULLType attribute. And when I changed RNULLType to NULLType, I get another error: tag "CB" not present, and the error came from the pysam library.

When I try to skip this step using either velocyto run or velocyto run-dropest, I get an error saying: the bam file does not contain cell and umi barcodes appropiately formated.

I tried multiple versions of rpy (3.5 and above, I did see the RNULLType attribute in the source code of older versions of rpy but was unable to install and test it), and velocyto 0.17.16 and 0.17.17.

As I was going through this (http://pklab.med.harvard.edu/velocyto/notebooks/R/SCG71.nb.html), it appears that one can get a velocity estimate from matricies of spliced and unspliced reads, which I can get from dropest directly. So I wonder if I can simply run the command gene.relative.velocity.estimate() inputting the matrix of spliced and unspliced reads from dropest directly? And what kind of difference should I expect from this compared to if I were to follow the tutorial thoroughly?

I am quite new to CLI and python so I apologize if my question seemed to be inappropriate at times.

Thank you in advance!

[ERICMIAO0817]

Hi, I wonder if your issue is solved or not, as a rookie in scRNA analysis, I have met some problems. Recently I am working on celseq2 data and wonder if velocyto is the correct tool for generating spliced and unspliced RNA data from bam file.Because of my limited knowledge, I bring up this naive question. Thank you in advance for your attention to this matter. I am looking forward to hearing from you soon.🥺

[bxxu]

Hi, I don't think I fixed the issue in the end. All I did was using the count matrix of spliced and unspliced mRNA generated by dropest to get velocity estimates using the velocyto R package.

As for whether velocyto is the right tool, I think it will depend on the velocity estimates you get in the end and whether it matches the biology of your system. Hope it's somewhat useful.

Hi, I wonder if your issue is solved or not, as a rookie in scRNA analysis, I have met some problems. Recently I am working on celseq2 data and wonder if velocyto is the correct tool for generating spliced and unspliced RNA data from bam file.Because of my limited knowledge, I bring up this naive question. Thank you in advance for your attention to this matter. I am looking forward to hearing from you soon.🥺

[ERICMIAO0817]

Thank you for your reply!!!Appreciate it.😃

[SevenTea7]

嗨， 我认为我最终没有解决这个问题。我所做的只是使用 dropest 生成的剪接和未剪接 mRNA 的计数矩阵，以使用 velocyto R 包获得速度估计。

至于 velocyto 是否是正确的工具，我认为这将取决于您最终获得的速度估计以及它是否符合您系统的生物学特性。希望它有点用处。

你好，不知道你的问题解决了吗，作为scRNA分析的菜鸟，我遇到了一些问题。最近我正在研究 celseq2 数据，想知道 velocyto 是否是从 bam 文件生成剪接和未剪接 RNA 数据的正确工具。由于我的知识有限，我提出了这个天真的问题。预先感谢您对此事的关注。我期待尽快收到您的来信。🥺

hello, I wonder if your problem has been solved? I ran into some problems when running velocyto. I hope you can help me. When I was using dropest, I was unable to install and use it correctly because some dependency packages were no longer available. Could you please share your dropest installer with me? Thank you in advance for your attention to this matter. I look forward to hearing from you soon.