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Old 02-11-2015, 01:56 PM   #8
n2hasan
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Location: Denver, CO

Join Date: Sep 2009
Posts: 6
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Greetings Jade,

It depends on the needs of your experimental design and if you were doing this yourself. If you are looking for species identification instead of genus and family level identifications of taxa, then the 16S Metagenomics Kit is your option. How many environmental samples/replicates per sample are you going to run? The protocol is very hands on/laborious compared to the standard microbiome sequencing, so it gets more cumbersome as you increase sample numbers that are common in microbiome experiments today. Also, what are you trying to do after identification of species in the sample? Comparing samples is something that is not included in the current IonReporter Metagenomics workflow.

The IonReporter workflow has two databases they analyze output against: the ThermoFisher MICROSEQ 16S Database that ThermoFisher manually curates, or the GreenGenes 16S Database. You can email ThermoFisher to check if certain groups of bacteria are included within the MICROSEQ database, but they are not willing to share those proprietary 16S sequences otherwise. They are also not willing to share the 16S primers used for the different V regions, so analyzing the data in other workflows (i.e. Qiime) is not as optimal as it could be.

The output of the run includes a graph visualizing the proprotions of different taxanomic levels within a sample and a sample specific table with % identity and read counts for taxonimic units (i.e. Family, Genus, Species, etc.) identified within a given sample. The table is not in biom format and requires some manipulation to get into the format for scripts included in Qiime. So doing analyses where you are comparing lots or samples will require some additional bioinformatic tool development on your end to do comparative analyses at this point.

For now, our lab mostly focuses on our custom V4 16S PGM sequencing and analyzing the data using Qiime. But if we are really trying to discriminate species in our clinical samples, we recommend the ThermoFisher 16S Metagenomics Kit.

You can try to have a few samples sequenced on a 314 or 316 chip for a pretty low cost. Microbiome sequencing doesn't need that many reads per sample (~200K per sample is the recommendation from ThermoFisher, but that always depends on how diverse your samples are.

I'm sorry if I didn't address what you were looking for, so feel free to ask specific questions.

Best Regards,

Nabeeh
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