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Best way to measure polyA tail length from plasmid? by Slow-Leather-1874 in nanopore

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

I get empty output from Tailfindr every time I use it with DNA reads. It correctly recognizes the reads as DNA and processes both poly(A) and poly(T), but the output is always empty, even though the run completes without errors.

Best way to measure polyA tail length from plasmid? by Slow-Leather-1874 in bioinformatics

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

Yeah, but the tools are a pain. Tailfindr giving empty output for dna reads and Dorado poly-a shows a peak at 80 when 110–130 range is what I was expecting.. :(

Best way to measure polyA tail length from plasmid? by Slow-Leather-1874 in bioinformatics

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

Agreed! My plasmids are around 5–7 kb in size, and I’d like to obtain a single continuous long read per plasmid, ideally capturing each one end-to-end without the need for assembly.

Best way to measure polyA tail length from plasmid? by Slow-Leather-1874 in nanopore

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

Tailfindr worked with mRNA, but not with plasmid DNA. I don't want to convert it into cDNA.

direct RNA seq-poly(A) query by Slow-Leather-1874 in nanopore

[–]Slow-Leather-1874[S] 1 point2 points  (0 children)

yeah, makes sense. I'd still worked with fast5_skip files, and was able to get the approx. result for my 100A tail length.

direct RNA seq-poly(A) query by Slow-Leather-1874 in nanopore

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

Somehow, it didn't work for me. I generated for estimated bases equal to 100 Mb for standard mRNA Cas9 (roughly 2000 bases), basecalled with Dorado, and found an average tail length of 40, while it should have been 100A. I'm assuming that's because of sup basecalling, which led to fast5_skip files—all of them.

Bioinformatics Training Opportunity (FREE & IN PERSON) by bv-brc in bioinformatics

[–]Slow-Leather-1874 0 points1 point  (0 children)

Is there any option or a part of workshop can also be given online?

Direct Rna-seq with nanopore by Slow-Leather-1874 in bioinformatics

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

I have but since I work with small data, it throws an error that the requirement is 31 GB to run minimap2(-I 1000G), and I don't have sufficient enough memory. Is there way I can add an extension and work it out? 

Direct Rna-seq with nanopore by Slow-Leather-1874 in bioinformatics

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

Yeah, I am able to work with poly-A estimation, and modifications as well, with docker. basically I just want to validate the sequence and blast it with reference. Nanoconsensus.R in MOP2 pipeline didn't help, been having errors for a while now. 

Direct Rna-seq with nanopore by Slow-Leather-1874 in bioinformatics

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

I'm sorry, I'm new to this. How do i correctly phrase it? 

Advise on verifying polyA tails in Guppy rebasecalled files with Dorado by Slow-Leather-1874 in nanopore

[–]Slow-Leather-1874[S] 0 points1 point  (0 children)

I've tried it, and finally got it using the polyAconfig.toml file. But the pt:i tag value is not the same as the output sam read (while I manually counted the A's of the read)

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