Any methods available to do QC analysis of Pacbio raw data??

Question

[karthic]

Hi,

I am very new to NGS. Am going to have pacbio raw data in few months. What am looking for is, methods to verify and validate the raw data. I want to find out how good is my data.

Please suggest approaches/methods/tools to do so.

TIA,

KK

[GenoMax]

Once the data is in fastq format you could use FastQC. That said you should use the SMRTlink tools that PacBio makes available to get the full benefit of information contained in raw data. If this is going to be a small number of runs you could just ask your sequence provide to provide you with the QC results from SMRTlink. If you are going to do this regularly then it may be useful to install the software locally.

[karthic]

Thanks genomax. Yes we are trying to install SMRTlink package. However my team wants to cross validate the data with third party tools. Particularly about max and avg read length obtained, library complexity and probably coverage. We have paid for ccs reads.But as per ur suggestion fastqc should help in covering some of them.

[colindaven]

I like the following for CCS reads

• alignment then visualization
• assembly if appropriate with Canu - which gives some great readout on the lengths, types and usefulness of the reads / library. The corrected reads which are output are also highly useful
• stats.sh from the bbmap package to get read length statistics.

As always, conda is very useful for installation.

[karthic]

Thanks colindaven. Sorry for delayed response as I was and lying in hospital. I will try your suggestions and I may pester you with more questions.

Answer 1

[WouterDeCoster]

If the name of the package doesn't offend your sequencer you could use my tool NanoPlot which can be used for extracting various metrics from fastq and bam files and plotting those. It's -as you might have guessed- written for Oxford Nanopore sequencing data but I can't think of a reason that it wouldn't work for PacBio.

[karthic]

Thanks WouterDeCoster. I will use your tool and share my experiences at the earliest.

Answer 2

[JstRoRR]

I think stsPlot is an R package dedicated for this purpose only. It plots primary analysis quality control metrics. https://github.com/PacificBiosciences/stsPlots

[Afif Elghraoui]

PacBio has since taken it down, but I had a clone of it and published it to keep it available: https://github.com/0xaf1f/stsPlots

Answer 3

[Rox]

A bit of an old post, but as I was still wondering about it today, I found this tool : LongQC . Never tried it yet, but I am going to !