PacBio SMRT command line Iso-seq analysis

Owned by Paul Whatmore (Deactivated)
Mar 15, 2023
2 min read

Email sent to Pawel Sadowski as part of Jira ticket https://eresearchqut.atlassian.net/browse/HSR-940

I wrote a basic guide for analysing Iso-Seq data generated by PacBio, using PacBio’s proprietary SMRT Link command line tools, as well as some additional community-developed tools.

 

Hi Pawel. Yes, the guide I wrote was for full-length 16S amplicon sequencing, but several of the processes are the same for Iso-seq, such as circular consensus sequencing, demultiplexing and primer removal.

But it might be better to start from the beginning.

The SMRTLink webserver is dead in the water, I’m afraid. It was extremely difficult to integrate it with the HPC to begin with (our devs had to end up editing the source code) but now with the extra security issues after the data breach, it’s a no-go.

You still can run all the tool via command-line though.

To install the current (v12) version of SMRT Link in your HPC home directory, follow this guide:

https://www.pacb.com/wp-content/uploads/SMRT_Link_Installation_v12.0.pdf

There’s a download link there as well.

On page 10 it shows you how to install just the command line tools (and not the full GUI package, which won’t work anyhow), see: “Installing only SMRT Tools” section. This will install all of the tools used by SMRT Link. The only difference is that they are run at the command line instead.

There’s a user guide here:

https://www.pacb.com/wp-content/uploads/SMRT_Link_User_Guide_v12.0.pdf

And a ref guide here that lists all of the tools installed and what they do:

https://www.pacb.com/wp-content/uploads/SMRT_Tools_Reference_Guide_v12.0.pdf

 

As for specific Iso-seq analysis guides using SMRT Tools, this website steps you through the whole process:

https://isoseq.how/getting-started.html

For each analysis section in this guide there is a ‘CLI (command-line interface) Workflow’ section that steps you though using the SMRT Tools command line tools.

 

For further downstream analysis, there are some community-developed tools recommended by PacBio:

https://github.com/GenomeRIK/tama

https://github.com/ConesaLab/SQANTI3

https://github.com/ConesaLab/tappAS

 

The SMRT Tools installation and command-line operations are pretty easy to install and use (no advanced Linux knowledge required). If you don’t have any command-line experience I can help you out getting it all set up. Give it a go anyhow, using the SMRT Link guides, and if you get stuck drop me an email.

 

Cheers

 

Paul

(eResearch Bioinformatician)