Pac Bio 18S reads

Theory behind mothur
1

Hi all,

Firstly I would like to say, thanks for developing mothur, looks like an incredible resource.

I’m going to preface my questions with the statement that I’m really new to working out how to deal with large sequencing datasets and have been working out how to use mothur - but I am a complete novice!

I have several 18S amplicon Pac Bio SMRT cell sequencing sets that I’ve been running through MG-RAST as a quick way to see what is there. However, I would really like to do some chimera detection and maybe have a go at analyzing my data without MG-RAST. I was wondering what your recommendations might be, perhaps someone has some experience of Pac Bio reads. I was thinking of following the 16S 454 SOP but any input would be greatly appreciated! I’m also happy to share my experience regarding the quality of data I’m getting out of the SMRT cell sequencing process.

Thanks for any help you may be able to give me,

Bethan

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Hey Bethan,

If you can sit tight for a few weeks we’ll have an SOP up for PacBio. It will be posted with the next release of mothur.

Pat

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Hi Pat,

Wow that sounds great! Thank you!

Bethan

PS I’d be interested to hear if anyone has had any issues with Pac Bio amplicon sequencing with regards to a) nucleotides being sheared off the 5’ or 3’ end during the formation of SMRTbells b) concatemers of amplicons being formed during library prep. I’ve had both of these situations come up which have caused issues with primers I had developed for barcoding. With respect to concatamers, I’ve been told that it is common for PacBio amplicon library prep - I’d be curious if anyone else had experienced this and how they dealt with it