Primers Product Of Length 1500 Bp

Question

[always_learning]

Hi All,

Can some one suggest platforms which can do sequencing for 1500 BP length primer product ? or if We know the maximum sequencing length from the one who will do the sequencing then We can designed primers accordingly. We are basically looking out for meta-genomic analysis here.

Any recommendations/Suggestions is welcome !!

Thanks

Syed

Answer 1

[Ido Tamir]

PacBio has a read length of more than 10kb, 50% longer than 5kb. 454 can now also up to 1kb.

http://en.wikipedia.org/wiki/DNA_sequencing#Next-generation_methods

Answer 2

[Josh Herr]

I'm a little confused here -- you're doing amplicon sequencing right? Let's be clear too -- using marker gene primers is not metagenomics. You did not give us any information about your primer region -- why you are using that region? ...and what type of mixed sample you are looking to sequence?

I don't think PacBio would be the best use of your resources in regards to amplicon sequencing from an environmental sample -- you'll get the read depth you need with another technique, such as Illumina (more, shorter reads) or 454 (few expensive reads, but up to 1000 bp in length). I don't know why you would need to sequence a marker at 1500 bp in length. Why do you need something at that length? If you MUST use that specific region for your research question, you should be fine with flanking marker regions.

PacBio is still getting the kinks out with their technique, but it's mainly optimal for longer sequencing projects (i.e. whole genomes) especially where you need to close gaps from repeats and hard to assemble regions. The read depth is still an issue and the error rate would wreck havok on any amplicon sequencing project from an environmental sample. So in other words -- even if you were willing to pay the PacBio money -- it would not be the correct technology choice for your research question.

[always_learning]

Hi Josh,

Thanks for detailed explanation !! As I am very novice in this field but will surely learn from you all.
This PRC products is a mix for thousands of microbes and its around 1500. So as per your suggestion shall we go for redesigning the primer so what should be maximum length so that we can meta-genomics in best (optimum) possible way ?

Answer 3

[bnbowman]

Syed,

It's true that PacBio is mostly used for genomic sequencing apps as Josh says, but recently we've been hard at work developing the platform for amplicon sequencing applications as well. Earlier this year I presented a poster on exactly this at ASM: http://files.pacb.com/pdf/Analysis_of_Full_Length_Metagenomic_16S_Genes_by_SMRT_Sequencing.pdf

Long story short, though our raw read accuracy requires some sophisticated algorithms to make the best use of, we can turn multiple passes over the same molecule into a high-quality "Circular-Consensus Sequence" which can mostly be analyzed with current tools. More over, with recent advances in sequence length we can now generate tens-of-thousands of reads of the length you desire from a single SMRTcell in ~2 hours. So if you're willing to put a bit more time and effort into the project on the analysis-side, I think our technology could suite your needs quite well.

Brett Bowman Scientist, Bioinformatics Pacific Biosciences, Inc.

[Josh Herr]

+1 on your answer, Brett. Sorry, didn't mean to rail into PacBio with my answer. I'm happy to see your poster and to know that you're working on this area. I'm excited to have access to a PacBio machine now, so I am hoping to give this a try in the near future. Thanks for your post and clarification!