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sheffield-bioinformatics-core / periscope

A tool to quantify sub-genomic RNA (sgRNA) expression in SARS-CoV-2 artic network amplicon nanopore sequencing data.
GNU General Public License v3.0
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illumina gRNA count #43

Closed pratibha-kadam closed 2 years ago

pratibha-kadam commented 2 years ago

Hi There, I have been working with Periscope on nanopore data but recently I tried to run the Periscopre on Illumina data. The output is not similar to that of ONT and the gRNA count is not provided. Can some one guide me why is it so? and how I can get the gRNA count?

Thank you Pratibha

DrownedMala commented 2 years ago

It was recently added in the latest version after discussion in the issue #41, you can install it following the instructions provided in the code page. It should be version 0.1.1.

Simone

pratibha-kadam commented 2 years ago

Thanks for the information but what I can see on the code page is version 0.1.0 instead of 0.1.1.

Is that 0.1.1 version not added yet?

Thanks and Regards Pratibha

On Tue, 5 Apr 2022 at 20:39, Mala @.***> wrote:

It was recently added in the latest version after discussion in the issue

41 https://github.com/sheffield-bioinformatics-core/periscope/issues/41,

you can install it following the instructions provided in the code page. It should be version 0.1.1.

Simone

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DrownedMala commented 2 years ago

I think they didn't upload the notes about the new version yet, but the script for Illumina reads was modified, so if you proceed git-cloning periscope now (and install it as a consequence), you will get gRNA count for Illumina too

Simone

pratibha-kadam commented 2 years ago

Thanks for the information.

To get the clarification on sgRNA/gRNA ratio, can you tell me is this a right way to calculate it?? I consider only high quality count for nanopore data and divide normalized count (RPHT) of all the gene's sgRNA except orf1a and divide it with gRPHT of orf1a sgRNA:gRNA = sgRPHT(S,ORF3a,E,M,ORF6,ORF7a,N,ORF10) / gRPHT(ORF1a)

if not can you guide me how to calculate it?

Many Thanks Pratibha

On Sat, 9 Apr 2022 at 14:14, Mala @.***> wrote:

I think they didn't upload the notes about the new version yet, but the script for Illumina reads was modified, so if you proceed git-cloning periscope now (and install it as a consequence), you will get gRNA count for Illumina too

Simone

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pratibha-kadam commented 2 years ago

Thanks for the information. To get the clarification on sgRNA/gRNA ratio, can you tell me is this a right way to calculate it?? I consider only high quality count for nanopore data and divide normalized count (RPHT) of all the gene's sgRNA except orf1a and divide it with gRPHT of orf1a sgRNA:gRNA = sgRPHT(S,ORF3a,E,M,ORF6,ORF7a,N,ORF10) / gRPHT(ORF1a) if not can you guide me how to calculate it? Many Thanks Pratibha … On Sat, 9 Apr 2022 at 14:14, Mala @.> wrote: I think they didn't upload the notes about the new version yet, but the script for Illumina reads was modified, so if you proceed git-cloning periscope now (and install it as a consequence), you will get gRNA count for Illumina too Simone — Reply to this email directly, view it on GitHub <#43 (comment)>, or unsubscribe https://github.com/notifications/unsubscribe-auth/AVDYCY66OWKNSTECP5BKXYLVEE7OHANCNFSM5SR237MA . You are receiving this because you authored the thread.Message ID: @.>

Can anyone guide me with this query??

Many Thanks Pratibha

mbdabrowska1 commented 2 years ago

I'm not exactly sure what is the goal of your calculation, so it's rather difficult for me to guide you on whether it is correct or not. Can you clarify what are you trying to show with this ratio?

pratibha-kadam commented 2 years ago

We are trying to look at the transcriptional activity in the host with sgRNA:gRNA ratio.

Thanks and Regards Pratibha

On Tue, 19 Apr 2022 at 16:29, Magda Dabrowska @.***> wrote:

I'm not exactly sure what is the goal of your calculation, so it's rather difficult for me to guide you on whether it is correct or not. Can you clarify what are you trying to show with this ratio?

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mbdabrowska1 commented 2 years ago

I suppose this calculation you mentioned above: sgRNA:gRNA = sgRPHT(S,ORF3a,E,M,ORF6,ORF7a,N,ORF10) / gRPHT(ORF1a) would work if you want to use ORF1a as your reference of transcriptional activity

I imagine dividing sgRPHT for all ORFs by gRPHT of all orfs (apart from ORF1a) could also be a solution: sgRNA:gRNA = sgRPHT(S,ORF3a,E,M,ORF6,ORF7a,N,ORF10) / gRPHT(S,ORF3a,E,M,ORF6,ORF7a,N,ORF10) since gRPHT values are available for each of these genes

As I mentioned, it is rather hard for me to tell exactly which of these solutions would be best in your case as it really depends what conclusions you are trying to draw from this comparison. Both of these approaches look at transcriptional activity from slightly different angles.