Introduction

[rsirois]

Hello everyone,

My name is Rachael and I am currently a second year Masters student at Smith College. I am pretty new to the NGS platform but I will be using the Illumina MiSeq for my thesis project. We will be using the MiSeq for a metagenomic project looking at the microbiome of the horse gastrointestinal tract. We will be using a 16s amplicon to assess the possible fluctuations in bacterial diversity and abundance in response to a pressure, namely treatment with a common antihelminthic drug.

[ScottC]

Hi.

I'd be interested to hear how well things go with your MiSeq and the 16S sequencing, if you're happy to share the information. Do you have an upgraded MiSeq (V2)? Is your lab already using it for 16S work, or are you starting it?

Cheers,

Scott.

[rsirois]

Hello Scott,

Thank you for your reply regarding the PCR Clean up kit. As far as the MiSeq, we have just received the sequencer about 3 weeks ago and so I am pretty sure it is the upgraded instrument. No one is the lab has had any experience with the MiSeq and so there are two projects that will be started. In addition to the 16S work (which I would be happy to let you know how it goes) one of our professors will also be using it to resequence her phage genomes. If you would like to read some articles about using the MiSeq platform for 16S analysis, the articles, Caporaso et al., 2011 (Global Patterns of 16S rRNA diversity) and Caporaso et al., 2012 (Ultra-high-throughput microbial community analysis) are very helpful. The latter is almost identical to what I would like to do with my samples.

[ScottC]

Hi,

Thanks for the link. I have seen those papers before. Just a warning, though... Some people seem to be getting poor quality results from the MiSeq when doing reduced complexity library sequencing (like 16S). So, speak to your FAS if it happens to you and they might be able to give some pointers.

All the best,

Scott.

[rsirois]

Hi Scott,

Thank you for the heads up. By reduced complexity library, are you referring to the fact that my library (since I am only doing 16S sequences) is not diverse? If so , the representative that came to install and train us on the MiSeq recommended that we may want to spike the library with up to 50% PhiX in order to generate some diversity.

Thanks,

Rachael

[ScottC]

Hi Rachael,

Yes, that's what I mean. I actually intended to say 'low complexity', not 'reduced complexity'!

Anyway, the PhiX spike may help, but you still may not get as good quality data as you do with other complex libraries like those produced by genomic DNA. There seem to be mixed reports as to how well they work. Some labs get good data, some don't. We've had mixed results. There's a bit of work going on in a few different labs to try to develop better methods for low-complexity library sequencing. Search for a few threads here on SeqAnswers and you'll find some information about the issues.

Regards,

Scott.

[guangxiwu]

thanks.....

[rsirois]

Hi Scott,

Thanks for the reply concerning the 16S low complexity problem. I'll be sure to look through some of the threads regarding this issue.

Best,

Rachael