= URL =

[http://biome.sdsu.edu/fastgroup/]

= Overview =

The FastGroup and FastGroupII programs were written by [http://phage.sdsu.edu Forest Rohwer]'s group at [http://www.sdsu.edu/ San Diego State University]. These programs were really written and tested on small 16S data sets typically from Sanger Sequencing. However, as is often the case, they work well for larger datasets like a single 454 run focussing on 16S genes.

FastGroup uses different clustering algorithms to generate both Richness (Chao1) and Diversity (Shannon-Wiener index) measures, as well as returning information about the groups and the statistics.

= Tips =

* I recommend removing the trimming options (by unchecking the boxes) unless you have a specific sequence you want to trim to.
* '''Please do not request the rarefaction curves''' These are computationally very expensive, and will take a long time to compute on large data sets.
* For Mac users, the calculation may take some time to compute and return. I recommend using firefox rather than safari. The latter times out after a few minutes whereas firefox keeps waiting.
* If you have problems with this site, please email the mg-rast mailing list. Although we do not control FastGroupII, we know the developers and will work with them. We are particularly concerned not to direct too much load on their server while we figure out a solution compatible with the mg-rast.

MG RAST Tutorial

2008-03-24T02:05:15Z

RobEdwards:

Papers that use the SEED

2008-01-08T06:18:44Z

RobEdwards: /* 2006 */

=2005=
# Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crécy-Lagard V, Diaz N, Disz T, Edwards R, Fonstein M, Frank ED, Gerdes S, Glass EM, Goesmann A, Hanson A, Iwata-Reuyl D, Jensen R, Jamshidi N, Krause L, Kubal M, Larsen N, Linke B, McHardy AC, Meyer F, Neuweger H, Olsen G, Olson R, Osterman A, Portnoy V, Pusch GD, Rodionov DA, Rückert C, Steiner J, Stevens R, Thiele I, Vassieva O, Ye Y, Zagnitko O, Vonstein V. (2005) Oct 7;33(17):5691-702. [http://www.ncbi.nlm.nih.gov/pubmed/16214803 PubMed] [http://nar.oxfordjournals.org/cgi/content/full/33/17/5691 NAR] [http://www.theseed.org/SubsystemPaperSupplementalMaterial/index.html Supplemental Online Material]
=2006=
# Angly, F.E., Felts, B., Breitbart, M., Salamon, P., Edwards, R.A., Carlson, C., Chan, A.M., Haynes, M., Kelley, S., Liu, H. et al. (2006) The marine viromes of four oceanic regions. PLoS Biol, 4. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=The%20marine%20viromes%20of%20four%20oceanic%20regions&cmd_current= PubMed] [http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0040368&ct=1 PLoS Biology]
# DeLong, E.F., Preston, C.M., Mincer, T., Rich, V., Hallam, S.J., Frigaard, N.U., Martinez, A., Sullivan, M.B., Edwards, R., Brito, B.R. et al. (2006) Community genomics among stratified microbial assemblages in the ocean's interior. Science, 311, 496-503. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=Community%20genomics%20among%20stratified%20microbial%20assemblages%20in%20the%20ocean%27s%20interior&cmd_current= PubMed]
# Edwards, R.A., Rodriguez-Brito, B., Wegley, L., Haynes, M., Breitbart, M., Peterson, D.M., Saar, M.O., Alexander, S., Alexander, E.C., Jr. and Rohwer, F. (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics, 7, 57. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=16549033&cmd_current= PubMed] [http://www.biomedcentral.com/1471-2164/7/57 BioMed Central]
# Gerdes, S., R. Edwards, M. Kubal, M. Fonstein, R. Stevens, and A. Osterman. (2006). Essential genes on metabolic maps. Curr Opin Biotechnol 17:448-56. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=16978855 PubMed] [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRV-4KWTFBB-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=3067cb821f4b494047d5270860f5557c Science Direct]
# Gerdes, S., Kurnasov, O., Shatalin, K., Polanuyer, B., Sloutsky, R., Vonstein, V., Overbeek, R., and A.L. Osterman (2006). Comparative Genomics of NAD Biosynthesis in Cyanobacteria. J. Bacteriol. 188: 3012-3023. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=16585762&cmd_current= PubMed] [http://jb.asm.org/cgi/content/abstract/188/8/3012 J. Bact]
# Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S., (2006). Experimental and computational assessment of conditionally essential genes in E. coli. J Bacteriol. 188(23):8259-71. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17012394 PubMed] [http://jb.asm.org/cgi/content/full/188/23/8259?view=long&pmid=17012394 JBact]
# Krause, L., Diaz, N.N., Bartels, D., Edwards, R.A., Puhler, A., Rohwer, F., Meyer, F. and Stoye, J. (2006) Finding novel genes in bacterial communities isolated from the environment. Bioinformatics, 22, e281. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=16873483&cmd_current= PubMed] [http://bioinformatics.oxfordjournals.org/cgi/reprint/22/14/e281 NAR]
# Rodriguez-Brito, B., Rohwer, F. and Edwards, R. (2006) An application of statistics to comparative metagenomics. BMC Bioinformatics, 7, 162.
# El Yacoubi B, Bonnett S, Anderson JN, Swairjo MA, Iwata-Reuyl D, de Crécy-Lagard V. (2006) Discovery of a new prokaryotic type I GTP cyclohydrolase family. J Biol Chem. Dec 8;281(49):37586-93. Epub 2006 Oct 10. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17032654&cmd_current= PubMed] [http://www.jbc.org/cgi/content/full/281/49/37586 JBC]# Fierer, N., Breitbart, M., Nulton, J., Salamon, P., Lozupone, P., Jones, R., Robeson, M., Edwards, R., Felts, B., Rayhawk, S. et al. (2007) Metagenomic and small-subunit RNA surveys reveal the high genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl Environ Microbiol, 73, 7059-7066. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17827313&cmd_current= PubMed] [http://aem.asm.org/cgi/content/full/73/21/7059?view=long&pmid=17827313 AEM]
# Yang, C., D. A. Rodionov, X. Li, O. N. Laikova, M. S. Gelfand, O. P. Zagnitko, M. F. Romine, A. Y. Obraztsova, K. H. Nealson, and A. L. Osterman. (2006). Comparative genomics and experimental characterization of N-acetylglucosamine utilization pathway of Shewanella oneidensis. J Biol Chem 281:29872-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=16857666 PubMed] [http://www.jbc.org/cgi/content/full/281/40/29872 JBC]

=2007=
# de Crecy-Lagard V, Hanson AD. (2007) Finding novel metabolic genes through plant-prokaryote phylogenomics. Trends Microbiol. Dec;15(12):563-70. Epub 2007 Nov 9. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17997099&cmd_current= PubMed] [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD0-4R3BW70-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=45c7be528111535816858125516a93ad Trends]
# de Crecy-Lagard V, El Yacoubi B, de la Garza RD, Noiriel A, Hanson AD. (2007) Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations. BMC Genomics. Jul 23;8:245.[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17645794&cmd_current= PubMed] [http://www.biomedcentral.com/1471-2164/8/245 Biomed Central]
# Gupta N, Tanner S, Jaitly N, Adkins JN, Lipton M, Edwards R, Romine M, Osterman A, Bafna V, Smith RD, Pevzner PA. (2007) Whole proteome analysis of post-translational modifications: Applications of mass-spectrometry for proteogenomic annotation.Genome Res. Sep;17(9):1362-77. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17690205 PubMed] [http://www.genome.org/cgi/content/full/17/9/1362 Genome Research]
# McNeil LK, Reich C, Aziz RK, Bartels D, Cohoon M, Disz T, Edwards RA, Gerdes SY, Hwang K, Kubal M, Margaryan GR, Meyer F, Mihalo W, Olsen GJ, Olson R, Osterman AL, Paarmann D, Paczian T, Parrello B, Pusch GD, Rodionov DA, Shi X, Vassieva O, Vonstein V, Zagnitko OP, Xia F, Zinner J, Overbeek R, Stevens R.(2007) The National Microbial Pathogen Database Resource (NMPDR): A genomics platform based on subsystem annotation. Nucleic Acids Res. 2007 Jan;35(Database issue):D347-53 [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17145713 PubMed] [http://nar.oxfordjournals.org/cgi/content/full/35/suppl_1/D347 NAR]
# Osterman, A., and T. Begley. (2007). A Subsystems-based approach to the identification of drug targets in bacterial pathogens, p. 132-170. In H.Boshoff and C.Barry (ed.), Progress in Drug Research, vol. 64. Birkhauser Verlag, Basel. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17195474 PubMed]
# Dmitry A. Rodionov, Oleg V. Kurnasov, Boguslaw Stec, Yan Wang, Mary F. Roberts, and Andrei L. Osterman (2007) Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate. . Proc Natl Acad Sci U S A. Mar 13;104(11):4279-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17360515 PubMed] [http://www.pnas.org/cgi/content/full/104/11/4279 PNAS]
# Wegley, L., Breitbart, M., Edwards, R. and Rohwer, F. (2007) Functional and taxonimic analysis of coral-associated microbes using metagenomic analysis. Environ Microbiol, 9, 2707-2719. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=17922755&cmd_current= PubMed] [http://www.blackwell-synergy.com/doi/abs/10.1111/j.1462-2920.2007.01383.x Environ Microbiol]
# Yang C, Rodionov DA, Rodionova IA, Li X, Osterman AL. (2007) Glycerate 2-kinase of Thermotoga maritima and genomic reconstruction of related metabolic pathways., J Bacteriol. 2007 Dec 21; [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&orig_db=PubMed&term=18156253&cmd_current= PubMed] [http://jb.asm.org/cgi/reprint/JB.01469-07v1?view=long&pmid=18156253 JBact]
# JR. Yates III and AL. Osterman (2007) Introduction: advances in genomics and proteomics. Chem Rev. Aug;107(8):3363-6. Epub 2007 Jul 21. [http://pubs3.acs.org/acs/journals/doilookup?in_doi=10.1021/cr068201u ACS]
=In press=
# Desnues CG, B Rodriguez-Brito, S Rayhawk, S Kelley, T Tran, M Haynes, H Liu, D Hall, FE Angly, RA Edwards, RB Thurber, P Reid, J Siefert, V Souza, DL Valentine, BK Swan, M Breitbart, F Rohwer (2008; accepted) Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature In press.
# Mou, X.S., S., Edwards, R.A., Hodson, R.E. and Moran, M.A. (2007) Generalist Species Dominate Bacterial Carbon Processing in the Coastal Ocean. Nature, In Press.
# Breitbart M, M Haynes, S Kelley, F Angly, R Edwards, B Felts, JM Mahaffy, J Mueller, J Nulton, S Rayhawk, B Rodriguez-Brito, P Salamon, F Rohwer (2008) Viral diversity and dynamics in an infant's gut.

2007-12-31T21:40:23Z

RobEdwards:

Papers that use the SEED

2007-12-31T19:30:29Z

RobEdwards:

RobEdwards: What files to upload

== File formats ==

To upload sequence data to the metagenomics RAST server, we accept several file formats.

* You can upload a fasta file containing ''just the nucleotide sequences''. This is the simplest format, just have a regular [[Valid fasta format]] nucleotide sequence file, and upload it. However, there may be some limitation on the file size.

* You can compress the sequence file containing ''just the nucleotide sequences'' with [http://www.gzip.org/ gzip], a popular compression tool. This will significantly reduce the size of the file to upload, and hence speed things up.

* You can also include a ''separate'' quality file in this same compressed file. To do this, compress both files into a single archive:

gzip archive.gz sequence.fa sequence.qual

and then upload the archive.gz file (don't worry, we'll take care of the name!)

If you do this, we will renumber the sequences and their corresponding quality scores at the same time. At the moment we don't use the quality scores, although we are experimenting with assembly tools that may take advantage of them. Therefore, the inclusion of quality scores is completely optional.

MG RAST Tutorial

2007-10-05T07:27:51Z

RobEdwards: /* Overview */

Which Sequences Should I Upload, and Where

2007-10-05T07:27:18Z

RobEdwards: /* Metagenomics Rast Server */

== Which Seqeunces Should I Upload? ==

We have different options for publicly available sequence analysis, and some can take different types of sequence data:

# The [[#Metagenomics Rast Server |Metagenomics RAST Server]] for metagenome sequences
# The [[#RAST Server|RAST Server]] for complete genomes

Please note, that most of our users problems are because the sequences are
# Not in [[Valid fasta format]]
# Not nucleotide sequences. At the moment we don't have any service for annotating just protein sequences. If there is a large demand, we can add this, but you probably don't want to use genome annotation tools to annotate protein sequences anyway.

=== Metagenomics Rast Server ===

The [http://metagenomics.nmpdr.org/ Metagenomics RAST Server] is designed to annotate '''nucleotide''' sequences from metagenome projects. You can supply either assembled or unassembled data, and reads can be as short as 100 bp and as long as you would like. There are some caveats to the system. Please also read this explanation of appropriate [[metagenomics sequence formats]].

==== Unassembled Data ====

If you want to do statistical comparisons between metagenomes, you most likely need unassembled sequences. The frequency that any gene is found is an approximation of the abundance of that gene in the environment. Thus if you two different samples you can compare gene frequencies between them to figure out which are the important environments. In this case, just upload the unassembled '''nucleotide''' sequences in [[Valid fasta format]]

==== Assembled Data ====

If you want to look for complete genes or pieces of a genome, then you can use assembled sequences. These are typically longer, and the ORF caller we use on the short fragments and sequences may have problems with longer sequences. On the ''to do list'' is to add specific ORF callers for different sequence sets.

For sequences over about 1,000,000 bp (1 Mbp) you should consider pulling out those sequences individually and running them through the [[#RAST Server|RAST Server]] server for complete genomes. This server uses far superior gene identification and analysis algorithms that are only applicable once you have longer sequences. However, the algorithm will not work very well with sequences under about 1 Mbp. If you assemble sequences you will loose the frequency information, and cannot easily do statistical comparisons between metagenomes.

=== RAST Server ===

The [http://www.nmpdr.org/anno-server RAST Server] is designed for complete, or nearly complete, microbial genomes. This uses a novel form of gene calling based on protein families, that is described in our upcoming paper. One of the basics of this technique is understanding where your organism lies using phylogenomics to ensure that we get accurate ORF calling. It doesn't make sense to use this technique for metagenomics. Furthermore, the RAST Server also leverages the SEED's work on functional coupling, assigning functions based on nearby genes.

The RAST Server is currently the best annotation platform for complete genomes.

== Remember! ==

Remember:
* '''nucleotides only'''
* '''[[Valid fasta format]]'''
* Metagenomes, assembled or unassembled, use the [http://metagenomics.nmpdr.org/ Metagenomics RAST Server]
* Complete genomes, or nearly complete (to the order of tens of contigs), use the [http://www.nmpdr.org/anno-server RAST Server]

MG RAST Tutorial

2007-10-05T07:26:20Z

RobEdwards: /* Overview */

Which Sequences Should I Upload, and Where

2007-10-04T19:18:25Z

RobEdwards: /* Which Seqeunces Should I Upload? */

== Which Seqeunces Should I Upload? ==

We have different options for publicly available sequence analysis, and some can take different types of sequence data:

# The [[#Metagenomics Rast Server |Metagenomics RAST Server]] for metagenome sequences
# The [[#RAST Server|RAST Server]] for complete genomes

Please note, that most of our users problems are because the sequences are
# Not in [[Valid fasta format]]
# Not nucleotide sequences. At the moment we don't have any service for annotating just protein sequences. If there is a large demand, we can add this, but you probably don't want to use genome annotation tools to annotate protein sequences anyway.

=== Metagenomics Rast Server ===

The [http://metagenomics.nmpdr.org/ Metagenomics RAST Server] is designed to annotate '''nucleotide''' sequences from metagenome projects. You can supply either assembled or unassembled data, and reads can be as short as 100 bp and as long as you would like. There are some caveats to the system:

==== Unassembled Data ====

If you want to do statistical comparisons between metagenomes, you most likely need unassembled sequences. The frequency that any gene is found is an approximation of the abundance of that gene in the environment. Thus if you two different samples you can compare gene frequencies between them to figure out which are the important environments. In this case, just upload the unassembled '''nucleotide''' sequences in [[Valid fasta format]]

==== Assembled Data ====

If you want to look for complete genes or pieces of a genome, then you can use assembled sequences. These are typically longer, and the ORF caller we use on the short fragments and sequences may have problems with longer sequences. On the ''to do list'' is to add specific ORF callers for different sequence sets.

For sequences over about 1,000,000 bp (1 Mbp) you should consider pulling out those sequences individually and running them through the [[#RAST Server|RAST Server]] server for complete genomes. This server uses far superior gene identification and analysis algorithms that are only applicable once you have longer sequences. However, the algorithm will not work very well with sequences under about 1 Mbp. If you assemble sequences you will loose the frequency information, and cannot easily do statistical comparisons between metagenomes.

=== RAST Server ===

The [http://www.nmpdr.org/anno-server RAST Server] is designed for complete, or nearly complete, microbial genomes. This uses a novel form of gene calling based on protein families, that is described in our upcoming paper. One of the basics of this technique is understanding where your organism lies using phylogenomics to ensure that we get accurate ORF calling. It doesn't make sense to use this technique for metagenomics. Furthermore, the RAST Server also leverages the SEED's work on functional coupling, assigning functions based on nearby genes.

The RAST Server is currently the best annotation platform for complete genomes.

== Remember! ==

Remember:
* '''nucleotides only'''
* '''[[Valid fasta format]]'''
* Metagenomes, assembled or unassembled, use the [http://metagenomics.nmpdr.org/ Metagenomics RAST Server]
* Complete genomes, or nearly complete (to the order of tens of contigs), use the [http://www.nmpdr.org/anno-server RAST Server]

Which Sequences Should I Upload, and Where

2007-10-04T19:16:48Z

RobEdwards: /* Remember! */

== Which Seqeunces Should I Upload? ==

We have different options for publicly available sequence analysis, and some can take different types of sequence data:

# The [[#Metagenomics Rast Server |Metagenomics RAST Server]] for metagenome sequences
# The [[#RAST Server|RAST Server]] for complete genomes

Please note, that most of our users problems are because the sequences are
# Not in [[Valid fasta format]]
# Not nucleotide sequences. At the moment we don't have any service for annotating just protein sequences. If there is a large demand, we can add this, but you probably don't want to use genome annotation tools to annotate protein sequnces anyway.

=== Metagenomics Rast Server ===

The [http://metagenomics.nmpdr.org/ Metagenomics RAST Server] is designed to annotate '''nucleotide''' sequences from metagenome projects. You can supply either assembled or unassembled data, and reads can be as short as 100 bp and as long as you would like. There are some caveats to the system:

==== Unassembled Data ====

If you want to do statistical comparisons between metagenomes, you most likely need unassembled sequences. The frequency that any gene is found is an approximation of the abundance of that gene in the environment. Thus if you two different samples you can compare gene frequencies between them to figure out which are the important environments. In this case, just upload the unassembled '''nucleotide''' sequences in [[Valid fasta format]]

==== Assembled Data ====

If you want to look for complete genes or pieces of a genome, then you can use assembled sequences. These are typically longer, and the ORF caller we use on the short fragments and sequences may have problems with longer sequences. On the ''to do list'' is to add specific ORF callers for different sequence sets.

For sequences over about 1,000,000 bp (1 Mbp) you should consider pulling out those sequences individually and running them through the [[#RAST Server|RAST Server]] server for complete genomes. This server uses far superior gene identification and analysis algorithms that are only applicable once you have longer sequences. However, the algorithm will not work very well with sequences under about 1 Mbp. If you assemble sequences you will loose the frequency information, and cannot easily do statistical comparisons between metagenomes.

=== RAST Server ===

The [http://www.nmpdr.org/anno-server RAST Server] is designed for complete, or nearly complete, microbial genomes. This uses a novel form of gene calling based on protein families, that is described in our upcoming paper. One of the basics of this technique is understanding where your organism lies using phylogenomics to ensure that we get accurate ORF calling. It doesn't make sense to use this technique for metagenomics. Furthermore, the RAST Server also leverages the SEED's work on functional coupling, assigning functions based on nearby genes.

The RAST Server is currently the best annotation platform for complete genomes.

== Remember! ==

Remember:
* '''nucleotides only'''
* '''[[Valid fasta format]]'''
* Metagenomes, assembled or unassembled, use the [http://metagenomics.nmpdr.org/ Metagenomics RAST Server]
* Complete genomes, or nearly complete (to the order of tens of contigs), use the [http://www.nmpdr.org/anno-server RAST Server]

Which Sequences Should I Upload, and Where

2007-10-04T19:16:05Z

RobEdwards: Which sequences should be uploaded to the RAST or MG-RAST services

== Which Seqeunces Should I Upload? ==

We have different options for publicly available sequence analysis, and some can take different types of sequence data:

# The [[#Metagenomics Rast Server |Metagenomics RAST Server]] for metagenome sequences
# The [[#RAST Server|RAST Server]] for complete genomes

Please note, that most of our users problems are because the sequences are
# Not in [[Valid fasta format]]
# Not nucleotide sequences. At the moment we don't have any service for annotating just protein sequences. If there is a large demand, we can add this, but you probably don't want to use genome annotation tools to annotate protein sequnces anyway.

=== Metagenomics Rast Server ===

The [http://metagenomics.nmpdr.org/ Metagenomics RAST Server] is designed to annotate '''nucleotide''' sequences from metagenome projects. You can supply either assembled or unassembled data, and reads can be as short as 100 bp and as long as you would like. There are some caveats to the system:

==== Unassembled Data ====

If you want to do statistical comparisons between metagenomes, you most likely need unassembled sequences. The frequency that any gene is found is an approximation of the abundance of that gene in the environment. Thus if you two different samples you can compare gene frequencies between them to figure out which are the important environments. In this case, just upload the unassembled '''nucleotide''' sequences in [[Valid fasta format]]

==== Assembled Data ====

If you want to look for complete genes or pieces of a genome, then you can use assembled sequences. These are typically longer, and the ORF caller we use on the short fragments and sequences may have problems with longer sequences. On the ''to do list'' is to add specific ORF callers for different sequence sets.

For sequences over about 1,000,000 bp (1 Mbp) you should consider pulling out those sequences individually and running them through the [[#RAST Server|RAST Server]] server for complete genomes. This server uses far superior gene identification and analysis algorithms that are only applicable once you have longer sequences. However, the algorithm will not work very well with sequences under about 1 Mbp. If you assemble sequences you will loose the frequency information, and cannot easily do statistical comparisons between metagenomes.

=== RAST Server ===

The [http://www.nmpdr.org/anno-server RAST Server] is designed for complete, or nearly complete, microbial genomes. This uses a novel form of gene calling based on protein families, that is described in our upcoming paper. One of the basics of this technique is understanding where your organism lies using phylogenomics to ensure that we get accurate ORF calling. It doesn't make sense to use this technique for metagenomics. Furthermore, the RAST Server also leverages the SEED's work on functional coupling, assigning functions based on nearby genes.

The RAST Server is currently the best annotation platform for complete genomes.

== Remember! ==

Remember:
* '''nucleotides only'''
* '''[Valid fasta format'''
* Metagenomes, assembled or unassembled, use the [http://metagenomics.nmpdr.org/ Metagenomics RAST Server]
* Complete genomes, or nearly complete (to the order of tens of contigs), use the [http://www.nmpdr.org/anno-server RAST Server]

MG RAST Tutorial

2007-10-04T18:56:57Z

RobEdwards: /* Overview */

Valid fasta format

2007-10-04T18:54:28Z

RobEdwards:

One of the most frequent errors with uploading the data is incorrect file format. We recommend fasta format for all the sequence data to be uploaded.

In particular, please check the following things:

# There should be no spaces or tabs at the start or ends of the lines
# The identifier line should begin with a greater than sign ">", and only one line is allowed
# Typically most bioinformatics applications use the first word after the > as the identifier for the sequence. Its nice (but not essential) if this is unique
# In the sequence lines (not header lines), spaces and numbers are removed.

Examples of valid fasta

>sequenceid
gatgcagcatgcagctagcagcgacggactac...
>1 this is a sequence that i know something about
gatgcagcatgcagctagcagcgacggactac...

Examples of invalid fasta

>sequenceid
This is a comment about the sequence
gatgcagcatgcagctagcagcgacggactac...
''Pleae don't include comments in the sequence data''

>sequenceid
gatgcagcatgcagctagcagcgacggactac...
''please don't have spaces before the > in the identifier''

fasta is probably the most common sequence format because it is relatively compact, and very easy to parse.

There is more information about the fasta format at:
# [http://en.wikipedia.org/wiki/Fasta_format Wikipedia]
# [http://www.ncbi.nlm.nih.gov/blast/fasta.shtml NCBI]

Valid fasta format

2007-10-04T18:53:58Z

RobEdwards:

One of the most frequent errors with uploading the data is incorrect file format. We recommend fasta format for all the sequence data to be uploaded. In particular, please check the following things:

# There should be no spaces or tabs at the start or ends of the lines
# The identifier line should begin with a greater than sign ">", and only one line is allowed
# Typically most bioinformatics applications use the first word after the > as the identifier for the sequence. Its nice (but not essential) if this is unique
# In the sequence lines (not header lines), spaces and numbers are removed.

Examples of valid fasta

>sequenceid
gatgcagcatgcagctagcagcgacggactac...
>1 this is a sequence that i know something about
gatgcagcatgcagctagcagcgacggactac...

Examples of invalid fasta

>sequenceid
This is a comment about the sequence
gatgcagcatgcagctagcagcgacggactac...
''Pleae don't include comments in the sequence data''

>sequenceid
gatgcagcatgcagctagcagcgacggactac...
''please don't have spaces before the > in the identifier''

fasta is probably the most common sequence format because it is relatively compact, and very easy to parse.

There is more information about the fasta format at:
# [http://en.wikipedia.org/wiki/Fasta_format Wikipedia]
# [http://www.ncbi.nlm.nih.gov/blast/fasta.shtml NCBI]

2006-08-30T16:48:53Z

RobEdwards:

=== Annotators SEED ===
The master copy for all data in the SEED environment. Users can not access this password protected site.
All annotations are made available via the [[#SEED-Viewer| SEED-Viewer]] and the [[#Trial-SEED|Trial-SEED]].

=== Annotation ===
please see [[#Assigning_a_gene_function_and_annotation| Assigning a gene function and annotation]]

=== Assigning a gene function and annotation ===
Annotators assign gene functions to genes, and we call this process annotation. In most contexts, people use the term annotation to refer to assignments of function to the genes within a single organism. We certainly use the term in this sense, but we also use it to describe the process of assigning functions to corresponding genes from numerous genomes. Our basic approach to annotation is to ask our annotators to annotate the genes included in a [[#subsystem|Subsystem]] (e.g., glycolysis) across all genomes. This process of annotation of the genes within a subsystem across a set of genomes, rather than annotation of genes within a single genome, allows our annotators to focus on a constrained set of functional roles and attempt to accurately identify exactly what variant, if any, of a subsystem exists in each of the genomes.

We use the term annotation to refer to assigning functions to genes (either within a single organism or to a constrained set of gene/protein families across a set of organisms). This activity certainly is closely related to the construction of subsystems and protein families (which we call FIGfams), but we will describe those activities elsewhere.

=== Assignment ===
please see [[#Assigning_a_gene_function_and_annotation| Assigning a gene function and annotation]]

=== Bidirectional Best Hit ===

The paper [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=EBI&pubmedid=10077608 The use of gene clusters to infer functional coupling]
defines a Bidirectional Best Hit or BBH as follows:

<blockquote>
Given two genes Xa and Xb from two genomes Ga and Gb, Xa and Xb are called a “bidirectional best hit (BBH)” if and only if recognizable similarity exists between them (in our case, we required fasta3 scores lower than 1.0 × 10−5), there is no gene Zb in Gb that is more similar than Xb is to Xa, and there is no gene Za in Ga that is more similar than Xa is to Xb. Genes (Xa, Ya) from Ga and (Xb, Yb) from Gb form a “pair of close bidirectional best hits (PCBBH)” if and only if Xa and Ya are close, Xb and Yb are close, Xa and Xb are a BBH, and Ya and Yb are a BBH.
</blockquote>

=== Clearinghouse ===
please see [[#SubsystemClearinghouse|Subsystem Clearinghouse]]

=== FIGfam ===
FIGfams are protein families generated by the Fellowship for Interpretation of Genomes (FIG). These families are based on the collection of subsystems, as well as correspondences between genes in closely related strains (we describe the construction of FIGfams in a separate SOP). The important properties of these families are as follows:

# Two PEGs which both occur within a single FIGfam are believed to have the same function.

# There is a decision procedure associated with the family which can be invoked to determine whether or not a gene can be “safely” assigned the function associated with the family.

=== Functional role ===
The concept of functional role is both basic and primitive in the sense that we will not pretend to offer a precise definition. It corresponds roughly to a single logical role that a gene or gene product may play in the operation of a cell.

=== Gene function ===
The function of a protein-encoding gene (PEG) is the functional role played by the product of the gene or an expression describing a set of roles played by the encoded protein. The operators used to construct expressions and the meanings associated with the operators are described in

http://www.nmpdr.org/FIG/Html/SEED_functions.html

Genes other than PEGs can also be assigned functions (e.g., SSU rRNA). However, in most cases the functions assigned to genes other than PEGs tend not to be problematic.

=== Metabolic Reconstruction ===
When we use the term metabolic reconstruction of a given genome we will simply mean the set of populated subsystems that contain the genome, the PEGs (and their assigned functions) that are connected to functional roles in these populated subsystems, and the specific variant code associated with the genome in each of the populated subsystems.

=== NMPDR pathogen genome ===

The NMPDR is responsible for five classes of genomes:

# Campylobacter jejuni
# Listeria monocytogenes
# Staphylococcus aureus
# Streptococcus pneumoniae and Streptococcus pyogenes
# Pathogenic Vibrio

=== PEG ===
A Protein Encoding Gene (PEG) is equivalent to a CDS (Coding Sequence).

=== Populated Subsystem ===
please see [[#Subsystem|Subsystem]]

=== SEED-Viewer ===
A read-only version of the SEED that presents the latest data.

http://seed-viewer.theseed.org

'''Please note''': The data is updated automatically every 24 hours. When citing or linking to the SEED please use this version.

=== Subsystem ===
A subsystem is a set of functional roles that an annotator has decided should be thought of as related. Frequently, subsystems represent the collection of
[[#Functional_role|functional roles]] that make up a metabolic pathway, a complex (e.g., the ribosome), or a class of proteins (e.g., two-component signal-transduction proteins within Staphylococcus aureus). A '''populated subsystem''' is a subsystem with an attached spreadsheet. The rows of the spreadsheet represent genomes and the columns represent the functional roles of the spreadsheet. Each cell contains the identifiers of genes from the corresponding genome the implement the specific functional role. That is, a populated subsystem specifies which genes implement instances of the subsystem in each of the genomes. The rows of a populated genome are assigned '''variant codes''' which describe which of a set of possible variants of the subsystem exist within each genome (special codes expressing a total absence of the subsystem or remaining uncertainty exist). Construction of a large set of curated populated subsystems is at the center of the NMPDR and SEED annotation efforts.

=== Subsystem Clearing House ===
Since annotators can work on any machine (including the public SEED) the way to propagate subsystems is via
http://clearinghouse.theseed.org/clearinghouse_browser.cgi?

=== Trial-SEED ===
A public, read-write copy of the SEED is made available on

http://theseed.uchicago.edu/FIG/index.cgi

'''Please note''': The data on this server is updated in irregular intervals. Users should not assume that annotations made on this system will persist. Please publish your annotations to the [[#Subsystem_clearing_house|Subsystem Clearing house]].

=== Variant Code===
please see [[#Subsystem|Subsystem]]