400bp chemistry 16s Amplicons

[AndrewThomas]

Dear all,

I would like to know if anyone has tried sequencing 16s rDNA amplicons that are in the correct size range (~320bp) using the Ion Plus Fragment Library Kit and One Touch System 2 then sequenced them using the new 400bp chemistry on a 314 v2 chip. Are there any incompatibilities? How does the data look? Even if they are not 16s amplicons, but other amplicons, I'm interested in your feedback.


Thanks in advance,



Andrew

[MrGuy]

I've been doing some work with amplicons in that range. Some mixed populations work, some not. Essentially the error profile is improved, you get the entire amplicon, reads drop off towards the end. It's all you pretty much expect in incremental improvments of the chemistry. If you go outside the specifications on the library size, you get no signal as expected.

edit: Because of the miseq carry over thread, I should also mention we have tested cross contamination and carry over pretty heavily and not really seen it as an issue resulting from instrumentation. I have noticed that my own barcodes (<1%) have more carry over than commercial types (undetectable) just because of the better design of the pattern the manufacturers use. I'm using those sequences in my own sythesis now.

[AndrewThomas]

Thanks for your feedback Mr.Guy!

I was a bit worried because there were some major changes in the base calling algorithms for the ion torrent server from version 1 to the latest version and I thought this could cave an even more predominant affect on longer reads... I know 454 signal processing had some issus with amplicons, especially 16S, due to the similaritiy between the sequences (lots of conserved regions), discussed in this blog post: http://pathogenomics.bham.ac.uk/blog...on-processing/

Do your reads get Q20 score on all bases untill de 400th nucleotide? And have you tried to increase the number of flows to get a little more sequence length (but not too much because of the decline in quality)?

[MrGuy]

We're in the early stages of the 400bp chemistry, so not too much to definitively say at this point. Regardless, we are looking into the similiarity issue and created something to look at reads as individual sequences skipping the idea of consensus. Filtering is done for Q20 for all our work. As for 400th nucleotide? Our amplicon was not that long (<350) but you get the entire read of amplicon. You won't get every molecule, but you will get enough to work with. You'll also get to see some fun oligosynthesis artifacts in the primer regions.

As a side note, our first service issue after 2 years has come up. Our OneTouch 2 was broken out of the box...

[MrGuy]

So, just ran a long amplicon. ~425 bp with 75% of reads over Q20 at the last base.