Hi there Mark, Thanks for the reply. So, the metagenome ID is 4465448.3 and its called Forno. The glitch has actually happened in others as well, and I'm only using this one as an example. I re-checked the numbers and they still don't add. I'm working with Genbank (e-5) for organism classification, and subsystems (e-5) for functional classification, for that matter. I have 209 hits in this entire metagenome for photosynthesis. If I check the number of photosynthesis hits among the bacterial hits I get 140, while the same for eukaryotic gives me 142 hits. That puzzled me. When I do the opposite, and check the taxonomic composition of just the 209 photosynthesis hits the numbers are even more weirder. I get 245 hits for bacteria and 403 for eukaryotes! Thanks again for the help, I'm available for any further explanations necessary, Best regards, Gustavo   Gustavo Bueno Gregoracci -- MSc., Dr. in Microbiology (Genetics and Molecular Biology)  Email: [email protected] -- Pos doc Student Laboratório de Microbiologia (Prof. Fabiano Thompson) Centro de Ciências de Saúde - Instituto de Biologia - UFRJ Tel: +55 (21) 2562-6567 

________________________________ From: Mark DSouza <[email protected]> To: Gustavo B. Gregoracci <[email protected]> Cc: MG- Rast <[email protected]> Sent: Monday, July 18, 2011 1:33 PM Subject: Re: Doubt about MG-Rast annotation

Hi,

Can you please send us the MG-RAST ID for the dataset where you observed this, we will check into it for you.

-- Regards, Mark D'Souza -- for the MG-RAST team

  [email protected]   http://metagenomics.anl.gov/

If you need to respond to this email please address it to the mailing-list [email protected]

----- Original Message -----

...

From: "Gustavo B. Gregoracci" <[email protected]> To: "Mark D'Souza" <[email protected]> Sent: Saturday, July 16, 2011 10:05:14 AM Subject: Doubt about MG-Rast annotation Hello there Mark,

As I was analyzing some metagenomes, I ran into a recurrent problem. I was trying to cross information from taxonomic and functional annotation, and discovered some inconsistencies. I could not figure those out so I’m writing you in the hope that you can help me, since this will affect my analysis.

You see… I wanted to understand the contribution of different phylogenetic groups to a given subsystem. So I got the total number of sequences for the photosynthesis subsystem, for example, which was 209 hits. Then I went into taxonomy and separated all entries identified as eukaryotes, sending them to the workbench. I changed again to functional and checked the total number of eukaryotic hits to photosynthesis, which was 163. So far so good, but I decided to perform the same analysis regarding prokaryotes. Sent all prokaryotic entries to the workbench and changed to functional to see their contribution to photosynthesis as well. I got 174 hits!

How is this possible? If my whole metagenome has 209 hits to a subsystem, how can I have 163 eukaryotic hits within it and 174 prokaryotic hits to the same subsystem? Can sequences be annotated as both eukaryotic and prokaryotic? Cause I was interpreting these categories as mutually exclusive…

Thanks in advance for the help, Looking forward to hear from you,

Best regards, Gustavo

Gustavo Bueno Gregoracci -- MSc., Dr. in Microbiology (Genetics and Molecular Biology) Email: [email protected]

-- Pos doc Student Laboratório de Microbiologia (Prof. Fabiano Thompson) Centro de Ciências de Saúde - Instituto de Biologia - UFRJ Tel: +55 (21) 2562-6567