10/26/13 Frequently Asked Questions - Metagenes

Frequently Asked Questions

From Metagenes

This FAQ presents in depth explanations of the bioinformatics analyzes necessary for the Annotathon. For a explanations on how to create
user accounts and general sequence management issues, please refer to the user manual, also called the Rule book (http://biologie.univ-
mrs.fr/annotathon/index.php?actionjs=rules) . Finally, please note this is a Wiki, so everyone is invited to contribute to this documentation!

Contents
1 Translation, ORFs and coding/non-coding status
2 INTERPRO: Identifying conserved protein domains
3 BLAST: Finding sequence homologs
4 Using BLAST to compile a list of FASTA formatted sequence homologs
5 A microbial view of the Tree Of Life
6 Designing sequence ingroups and outgroups for phylogenetic tree inference
6.1 Strategy for defining ingroups and outgroups
6.2 List of complete microbial genomes at NCBI
6.3 Common pitfalls & difficulties in building trees
7 Phylogeny.fr: Infering phylogenetic trees

Translation, ORFs and coding/non-coding status

1. If the genomic DNA does not contain any Open Reading Frames (ORFs, here defined as a stretch of at least 60 codons without a
single STOP codon), then immediately conclude NON-CODING (tick non-coding under STATUS). Other than a very brief word
of conclusion, no other analyses or annotations are required (non coding DNA annotation is difficult with very short random
metagenomic DNA reads).

1. If the genomic DNA does contain ORFs over 60aa in length, proceed with the rest of the analysis with the longest available
ORF. Two outcomes are possible:
analysis of the longest ORF shows homologs and/or conserved protein domains => select coding STATUS and proceed
with the rest of the analyzes (multiple alignements, phylogeny etc.)
analysis of the longest ORF shows no homologs (even in the ENV_NR environmental sequence database) and no
conserved protein domains => discuss if the DNA is coding or not and select the appropriate STATUS. If the ORF is very
long (say over 200aa), then it is likely that this ORF does indeed code for a protein: it is then called an ORFan - an ORF
with no known homologs! If the ORFan is only just above the 60aa length threshold, you might want to classify it as non-
coding. Also beware of low complexity DNA (e.g. repeated stretches of the same bases), as this is often found to yield long
false positive ORFs (in which case the translations usually also bear highly prominent AA repeats). In any case, discuss
your choice and always carry out a BLASTx before concluding that the DNA is non-coding. Only proceed with the analysis
of a lesser sized ORF, if it is largely overlapping with a longer ORFan and shows BLAST homologs or conserved protein
domains. This is not so common but has been seen a few times in the GOS data (the real ORF, with clear homology to
known proteins, is contained in a larger false positive ORF with no matches, usually antisense).

As far as what inititation codon parameter to select in the ORF finding software, start with the greedy approach: that which produces the
longest possible ORFs (i.e. use "any codon" for ORF start in SMS/Orfinder). If later on your multiple alignments seem to suggest that all
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your homologs start further

downstream, then revisit the
ORF start position by locating its
most likely start codon (the one
closest in position to the
homolog's starts).

In terms of what genetic code to

use for generating the ORF,
select either the
"universal/standard code", or the
one most likely used by the hosts
of your DNA fragments
("bacterial" for marine samples
passed through .8 micron filters).

If you use SMS/ORFinder,

remember to carry out the
analysis in all 6 frames! Frames
1, 2 & 3 on direct then
indirectstrand.

INTERPRO:
Identifying
conserved protein
domains
ORF finding
Identifying conserved protein
domains in a protein is a
powerful method to predict its putative function.

Paste your protein sequence in the sequence field of the InterproScan (http://www.ebi.ac.uk/InterProScan/) tool: click "Submit Job" to
scan your protein against the large InterPro (http://www.ebi.ac.uk/interpro/) federation of protein domain databases.

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InterProScan home page

You might have to wait a few

minutes for the results to be
returned. In the resulting list of
predicted InterPro domains, take
note of the following points:

an InterPro record (e.g.

IPR000165) corresponds
to a number of identified
conserved domains from
the underlying databases
(here domain PR00736
from PRINTS &
PS00820 from
PROSITE); please
indicate in the
corresponding
Annotathon field the Extract from the results page
InterPro Accession
Number (here
IPR000165)
all the InterPro domains identified in your sequence are not necessarily independent: some domains might be contained in others, or
can have child/parent relationships. Click on "Table View" at the top of the results page to obtain a more detailed output (including
domain start & stop positions, as well as the all important E-values associated with the predictions)
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ignore any InterPro domains that are tagged as "Unintegrated", unless you have absolutely nothing else to feed your "functional
role" line of investigation
click on the "Raw output" button to see the full results in the "text only" format, suitable for copying & pasting into the "Domains"
raw results section of the Annotathon (copy the results in extenso, not just the domains your consider of interest)

You can see which other

domains are linked to the
domains identified in sequence in
the "Children"/"contains"/"found
in" sections of the InterPro scan
results. Rule: only define a
specific domain in the
Annotathon for the largest
encompassing domain, i.e. the
domain which contains the
other ones.

In this example, the first domain

(IPR000165) has domains
IPR008291 as children (4th in
the results list); in this case only
indicate IPR000165, and not
IPR008291.

It is also in the Table View that

you will find the exact
coordinates of the predicted
domains; for domain
IPR000165, report the
extremities of the PROSITE
domain, and not those of all the
small PRINTS sub-fragments! Table View of same results

Note that in some InterPro

records, you will find precious
functional hints for the conserved
domains. You can use these
functional indications to help you
select appropriate Gene
Ontology terms for your protein
(Molecular Function and/or
Biological Process). Sometimes,
specific GO terms are in fact
directly associated with InterPro
domains, which might prove very
useful if you think these GO
annotations can be transfered to
your particular protein.

Example of fileld out Annotathon conserved domains section

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BLAST: Finding sequence homologs

Search for known protein
sequences that look similar to
your ORF (potential homologs

Figure B1: NCBI BLAST submission form

(http://en.wikipedia.org/wiki/Homology_(biology)) ) by running BLAST, preferably using the NCBI online BLAST

(http://www.ncbi.nlm.nih.gov/BLAST/) (since it presents the all important Taxonomy Report), or at other institutions offering online
BLASTs (e.g. the EBI (http://www.ebi.ac.uk/blast/) , or GigaBlaster@IGS (http://www.igs.cnrs-mrs.fr/Giga2/~database/remoteblast.cgi)
).

The most usual BLASTs for the Annotathon are:

BLASTp versus SWISSPROT: to find homologs that are well annotated (e.g. molecular functions etc.)
BLASTp versus NR: find all possible homologs (e.g. to carry out a phylogenetic analysis)
BLASTx versus NR: translates your genomic fragment directly into the 6 possible frames and then runs 6 BLASTp's (if you are
unsure of the ORF location, or if you suspect sequencing errors producing frameshifts)

Start by filling out the BLAST online form(Fig. B1):

copy/paste your query sequence (ORF protein sequence for BLASTp, full genomic DNA sequence for BLASTx)
select the databank you wish to search: usually SWISSPROT or NR (NR is a compilation of all protein databanks, and therefore

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contains all known protein sequences)

select a higher number of Max target sequences than the default 100 (say 1000, sometimes more) in order to get the full
spectrum of homologs. If you don't select a high enough number of target sequences, then your list of similar sequences might end up
truncated (you will know this is the case when the bottom of your resulting list of BLAST hits doesn't reach the default E-value
threshold of 10).

After having submitted the

search, wait until the 'BLAST
Status ... Searching' page (Fig.
B2) is finally replaced by the
results page. Note, however,
that this 'BLAST Status ...
Searching' intermediate page can
sometimes present a colored
diagram which corresponds to a
conserved domain search result
(in this case against the CDD
domain database). This can
prove very useful (see above),
but has nothing to do with the
BLAST results per se!
Figure B2: Screenshot of the NCBI 'BLAST Status ... Searching' self-refreshing page

For a BLAST result which you

wish to report in the Annotathon,
please always include in the
Annotathon BLAST section
(Fig. B6):

a header/protocol line
which non-ambiguously
describes what search
was carried out (ex:
BLASTp versus
SWISSPROT, NCBI
default parameters other
than "500 max target
sequences")
the full, unabridged, list of
hits and E-values (Fig.
B4)
the first dozen pairwise
alignments only (Fig. B5)
the full, unabridged,
Figure B3: BLAST results header
taxonomic report (the first
section, entitled Lineage
Report Fig B7) copied into the Annotathon "Taxonomic Report" section (Fig B8)

NCBI graphical overview of pairwise alignments found

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Figure B3: NCBI graphical overview of pairwise alignments found

List of BLAST hits and E-values

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Figure B4: list of BLAST hits and E-values

List of detailed BLAST pairwise alignments

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Figure B5: list of detailed BLAST pairwise alignments

Annotathon: BLAST results section

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Figure B6: Annotathon: BLAST results section

NCBI BLAST "Taxonomic

Report" (Lineage report)

Figure B7: NCBI BLAST "Taxonomic Report" (Lineage report)

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If you wish to report more than

one BLAST results in the
Annotathon (e.g. one vs
SWISSPROT & one versus
NR), copy them one after the
other in the Annotathon field with
a line of dashes as a separator(--
---------------------------).

Figure B8: Annotathon section for BLAST "Taxonomic Report" (Lineage report)

Using BLAST to compile a list of FASTA formatted sequence homologs

Take advantage of your fresh BLAST main results page to compile a set a FASTA formatted sequences (e.g. your in group and out group
sequences to carry over to phylogenetic analysis).

Go to the pairwise alignments section of your NCBI BLAST report, and follow instructions in the following screenshots.

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A microbial view of the Tree Of Life

It is essential that metagenomic sequence annotators keep this simplified Tree of Life within reach at all time! Understanding the branching
patterns is quintessential to correctly define in- and out-groups for infering phylogenetic trees. You could print out the image below, or
make it your Desktop background image...

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Designing sequence ingroups and outgroups for phylogenetic tree inference

Strategy for defining ingroups and outgroups

1. Define the ingroup so that it represents a true taxonomic lineage

It must be a monophyletic group!
Choose a wide enough range of sequences so that all ingroup lineages are represented
2. Define the outgroup as the set of all other lineages of the same taxonomic level as the ingroup
Choose a wide enough range of sequences so that all outgroup lineages are represented
If you have no available sequence homologs for outgroup species, then you will have no outgroup (the tree will be unrooted)

Above all, remember that each and every sequence you wish to include in your phylogenetic tree should be a clear homolog of the others:
each sequence should have a credible BLAST E-value when aligned to your query, and each sequence must fit snuggly in the multiple
sequence alignment! Any sequence that looks like it doesn't belong to the same family, or is too partial (truncated) compared to other
members of the family, should be removed from the in or out groups!

Valid examples of ingroups (always refer to the Tree Of Life):

Cyanobacteria
Thermotogales
delta-Proteobacteria
Planctomycetales + Chlamidiales + Verrucomicrobiales (PVC)
Proteobacteria
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beta-Proteobacteria +gamma-Proteobacteria
Bacteria
Archaea
Cellular organisms (Archaea + Bacteria + Eukaryotes)

Invalid ingroups:

Cyanobacteria + Firmicutes
alpha-Proteobacteria + beta-Proteobacteria

Example n°1:

Ingroup = Firmicutes
Outgroup = other lineages of same taxonomic level (i.e. all
other bacterial phyla: Thermotogales, Aquificales,
Cyanobacteria, Proteobacteria, PVC...)
Your groups should contain representatives of each of
Microbial Tree Of Life
these phyla

Example n°2:

Ingroup = alpha-Proteobacteria
Outgroup = other lineages of same taxonomic level (i.e. all other Proteobacteria: beta, delta, epsilon, & gamma)
Your groups should contain representatives of each of these classes

Example n°3:

Ingroup = gamma-Proteobacteria
Outgroup = other lineage of same taxonomic level (i.e. beta-Proteobacteria)
Your groups should contain representatives of each of these two classes

Example n°4:

Ingroup = Bacteria
Outgroup = other lineages of same taxonomic level (i.e. Archae & Eukaryotes)
Your groups should contain representatives of each of these domains

Example n°5:

Ingroup = Bacteria
If there are no archeal or eukaryotic homologs, you will not use an outgroup (the resulting tree will be unrooted!)

Important: it is essential that you select the sequences to build the in and out groups in such a way that these group's full diversities are
well represented (i.e. that you have sequence representatives of each of the subgroups that make up the in and out groups). Use the above
simplified Tree Of Life, the NCBI Taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) and the BLAST
Lineage Report to identify existing subgroups.

Example: In the tree opposite, the in group is made up of the pink and blue branches, the unknown query sequence is highlighted in
yellow. The out group is made of the green and red branches.

for the in group, pick 15-30 sequences in each one of the subgroups enclosed by a pink or blue bracket (e.g. those on a grey
background)
for the out group, pick 5-10 sequences in each one of the subgroups enclosed by a red or green bracket (e.g. those on a grey
background)
in the example across, the resulting phylogeny would successfully suggest that the query sequence belongs to the pink group,
probably even to the same subgroup as Nitrosomonas europaea
under no circumstance should you just pick the set of first 15 best BLAST scoring hits for you in (or out) group! This will usually
result in just representing a single subgroup...

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List of complete microbial genomes at NCBI

To the right is a table of Bacteria

complete microbial genomes
are available at the NCBI Gamma-Proteobacteria 145
(2007): Firmicutes 129
Alpha-Proteobacteria 79
Of course, many more partial
genome sequences from other Beta-Proteobacteria 48
bacteria or archae are present Actinobacteria 48
in GENBANK or
SWISSPROT. However, if a Cyanobacteria 30
study conducted with a Epsilon-Proteobacteria 19
gamma-proteobacterial in Delta-Proteobacteria 18
group reveals only a handful
gamma-proteobacteria, then Bacteroidetes/Chlorobi 17
uttermost care is required PVC 13
during interpretation. Indeed,
Spirochaetes 9
since over 145 complete
gamma-bacterial genomes are Chloroflexi 8
available in GENBANK, this Thermotogales 6
might indicate horizontal gene
transfers, or massive gene Thermus/Deinococci 4
loss in this group. Acidobacteria 2
Aquificales 1
Other 2
Archaea
Euryarchaeota 33
Crenarchaeota 15
Nanoarchaeota 1

Selecting sequences representing the full diversity range

Common pitfalls & difficulties in building trees

Pitfall 1: in group sequences do not fully represent group diversity

In the figure across, the in group is made of the pink + blue groups, the query sequence is highlighted in yellow, and the out group is made
of the red + green groups.

An incorrect selection of in group sequences is indicated by the light grey backgrounds. The resulting inferred phylogeny (right panel) will
show the query sequence emerging exactly between the in group and the out group. This usually indicates :

an incorrect selection of sequences to represent the in group (and/or out group).

an incorrect definition of the in group (and/or out group)
Solution 1: select a more rational set of sequences that better represents the in group

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Pitfall 1: in group sequences do not fully represent group diversity

Solution 2: redefine the in and out groups

If no amount of wider ranging in group sequence selection manages to integrate the query sequence, then this might be a true
biological signal rather than an artefact (see below). This does arise occasionally when dealing with metagenomes since these
sequences can come from uncultured bugs belonging to potentially never seen before taxonomic subgroups (i.e. discovery?).

Difficulty 1: query sequence never integrates the in group

In the figure below, the in group is blue, the outgroup pink, and the query sequence yellow (infered phylogeny n°1). Regardless of the
efforts to properly represent the in group diversity, the query sequence always emerges between the in and out groups. Left in this state, no
conclusion is possible from the infered phylogeny n°1.

Solution : broaden the out group (add further green and red out groups) and rerun tree inference. Two outcomes are possible using
the broadened outgroup:
1. the query sequence is specifically linked to the in group (without integrating the latter, inferred phylogeny n°2): it is
legitimate to conclude that the query sequence is a close relative of the in group, even if one can not conclusively state that it is
part of it. The query sequence represents either an unknown subgroup of the in group, or it represents an unknown novel
group, close relative of the in group

1. the query sequence is not specifically linked to the in group in particular (inferred phylogeny n°3): it is legitimate to
conclude that the query sequence represents an unknown altogether novel group, not specifically related to the in group

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Difficulty 1: query sequence never integrates the in group

Difficulty 2: anomalous classifications of in and out

group sequences (HGT's)

In the figure opposite, sequence classification

according to the inferred phylogeny presents
occasional contradictions with the accepted
reference phylogeny (Tree of Life). Some in group
sequences are mixed in the out group branch,
and/or outgroup sequences are mixed within the in
group branch. Less dramatic anomalies occur when
in and outgroup sequences are well separated, but
mixes occur between lineages within either the in
group or the outgroup.

Explanation : the sequence is likely to be

subject to horizontal gene transfers
(HGT's, some genes are more frequently
observed in HGT's, such as antibiotic
resistance genes and various transporters).
In the figure opposite, we can only conclude
that the sequence is a close relative of
Ralstonia solanacearum, without it being
possible to assign the query to either pink or
blue groups.

Difficulty 2: anomalous classifications of in and out group sequences

Difficulty 3: anomalous classifications of in and out group sequences (duplications)

In the figure below, the conventional species phylogeny is shown on the left (True phylogeny). The phylogeny inferred from a set of
homologous sequences is shown in the center (Inferred phylogeny), and shows an additional red branch linked specifically to the blue
branch. This unexpected inferred phylogeny can be explained by either:

Gene duplication followed by differential losses in various lineages (right panel)

Horizontal gene transfer from the blue branch to some members of the red group (bottom panel)

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Resolving past duplication events is notoriously difficult; it usually involves restricting the analysis to species for which a complete genome
sequence is available, allowing the inference of trees containing all paralogs and orthologs involved. However, differential gene loss which
often follows gene duplications can make inferred trees rather cryptic...

Difficulty 3: anomalous classifications of in and out group sequences (duplications)

Phylogeny.fr: Infering phylogenetic trees

With your in-group and out-group set of FASTA formatted sequences, point your browser to the http://www.phylogeny.fr/ online site for
multiple sequence alignment and phylogenetic tree construction. You will find below a screenshot tutorial of the full procedure:

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www.phylogeny.fr home page

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Workflow setup

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Data entry

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Multiple alignment

The "MUSCLE" format alignment obtained by clicking the "Alignment in CLUSTAL format" link (paste in Annotathon multiple
alignment field): ):

MUSCLE (3.7) multiple sequence alignment

gi|8613437 ------------------MSNSRKRHEALLYHAKPKPGKIAVVPTKKYATQHDLALAYSP
GOS_26940 ------------------------------------------------------------
gi|8870682 --------------MDDDKSRQAARDAALRYHAYPKPGKLEIRATKPLANGQDLARAYSP
gi|2066869 -----------------MSDSQNLRQAALNYHEFPRPGKLEIRATKPMANGRDLARAYSP
Spomeroyi -----------------MSDQPSLRQAALDYHAFPKPGKLEIRATKPMANGRDLARAYSP
gi|1584252 ----------------MSNISEDLKSGALVYHRSPKPGKLEIQATKPLGNQRDLALAYSP
gi|1529713 -------------------MDEQLKQSALDFHEFPVPGKIQVSPTKPLATQRDLALAYSP
gi|7680888 ----------MSTSSSSSSSKEKLREAALDYHEFPTPGKVAIAPTKQMINQRDLALAYSP

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gi|1879253 MPSNVYSNPPSEARLMSTPVNSKLREAALDYHEFPTPGKIAIAPTKQMINQRDLALAYSP

gi|8613437 GVAEPCLEIAKDKNNIYKYTSKGNLVAVISNGTAVLGLGDIGPEASKPVMEGKGLLFKIF
GOS_26940 ------------------------------------------------------------
gi|8870682 GVAEACLEIVKDPATAADYTARGNLVAVISNGSAVLGLGNIGGLAAKPVMEGKAVLFKNF
gi|2066869 GVAEACTEIQADAANAARYTSRGNLVAVVSNGSAVLGLGNIGALASKPVMEGKAVLFKNF
Spomeroyi GVAEACLEIKDNAAHAETYTARGNLVAVVSNGTAVLGLGNIGALASKPVMEGKAVLFKKF
gi|1584252 GVAAACEAIKADPLQAAELTTRANLVAVVSNGTAVLGLGNIGPLASKPVMEGKAVLFKKF
gi|1529713 GVAAPCLEIEKDPLAAYKYTARGNLVAVVSNGTAVLGLGNIGALAGKPVMEGKGVLFKKF
gi|7680888 GVAFACEEIVENPLNAARFTARSNLVGVVTNGTAVLGLGNIGPLASKPVMEGKAVLFKKF
gi|1879253 GVAFACEEIVENPLNAARFTARSNLVGVVTNGTAVLGLGNIGPLASKPVMEGKAVLFKKF

gi|8613437 AMKLAAVHALADLAKKSVPEQVNIVYDEVSLNFGKEYIIPKPFDPRLIYEIPPAVAKAAM
GOS_26940 -----------------------------------------PFDPRLSSVVSSAVAEAAM
gi|8870682 AMQLACIDGIAALSRATTSAEAAEAYRGEQLVFGVDYLIPKPFDPRLMGVVASAVASAAM
gi|2066869 EMQIACVDGIAELARATTSAEAAAAYKGEQLNFGADYLIPKPFDPRLVAVVSSAVAKAAM
Spomeroyi AMQIACVEGIAELARITTSAEAAAAYQGEQLTFGADYLIPKPFDPRLVGVVSSAVARAAM
gi|1584252 EMKMAAVEAIAALARETPSDVVARAYGGETRAFGADSIIPSPFDPRLILRIAPAVAKAAM
gi|1529713 EMKLAAVHAIAELAHAEQSEVVASAYGDQDLSFGPEYIIPKPFDPRLIVKIAPAVAKAAM
gi|7680888 EMEIAAVNAIAELAQQEQSDIVATAYGIQDLSFGPEYLIPKPFDPRLIVKIAPAVAQAAM
gi|1879253 EMEIAAVNAIAELARQEQSDIVATAYGIQDLSFGPEYLIPKPFDPRLIVKVAPAVAKAAM
****** :..*** ***

gi|8613437 ESGVALEPISDWDAYREELMERSGSGSKEIRQIHNRAK---RNKKRIVFAEADHLDVLKA
GOS_26940 QSGVATQPIKDIDAYRDALKQTVVKSAFLMRPVFEAAS---SSARRIVFAEGEDERVLRA
gi|8870682 ETGVATRPVEDLVAYRERLDASVFRSSMIMRPVFAAAA---LSQRRIVFAEGEDERVLRT
gi|2066869 ESGVATRPIEDITAYKQKLNQTVFKSALLMRPVFEAAR---AAARRIVFAEGEDERVLRA
Spomeroyi ESGVARRPITDLEAYRQKLNQSVFKSALLMRPVFEAAA---KAARRLVFAEGEDERVLRA
gi|1584252 DTGVATRPIADFDAYNEKLDEFVFRSGFIMRPLFQRAK---QDKKRVIYAEGEDERVLRA
gi|1529713 DSGVATRPIADFDAYIEKLSEFVYKTNLFMKPIFSQAR---KEPKRVVLAEGEETRVLHA
gi|7680888 DGGVATRPIEDMEAYKVHLQQFVYHSGTTMKPVFQIARGAPAEKKRVVFAEGEEERVLRA
gi|1879253 DSGVAERPIEDMEAYEQHLQQFVYHSGTTMKPIFQLARGVEPEKKRIVFAEGEEERVLRA
: *** *: * ** * :. :. * .*:: **.: **.:

gi|8613437 AQRVQEEKLGLPILLGRKEVILELKEEIGFT----EDVPIFDPKTDEEKERRDRFGIAYW
GOS_26940 AQAVLEETSEVPIVIGRPEVIQQRCERLGLDIRPDRDFNIVNPQQD---DRYRDYWTSYH
gi|8870682 AQVIVEEMTDRPILIGRPEIIARRCEKAGLTIKPGEDFEVVNPEDD---SRHRRYWEAYL
gi|2066869 AQAILEETTETPILIGRPEVIERRCEKLGLDVRPGRDFQLVNPEND---PRYYDYWNSYH
Spomeroyi AQAILEETTETPILIGRPEVIEARCEKMGLSVRPGQDFQIVNPEND---PRYYDYWTSYH
gi|1584252 AQAVIEEGIAHPILVARPSVLEARLQRFGLSIRPGKDFEVINPEDD---PRYRDFVRSYI
gi|1529713 TQELVSLGLAKPILVGRPSVIEMRIQKLGLQIKAGVDFEIVNNESD---PRFKEYWSEYY
gi|7680888 VQIVVDEKLAKPILIGRPAVIEHRIQRYGLRLTPGVDFTIVNTEHD---ERYRDFWQTYF
gi|1879253 MQIIVDEKLAKPILIGRPAVIEQRIARYGLRLIAGQDYTVVNTDHD---ERYRDFWQEYH
*:. **::.* :: *: * :.: . * * : *

gi|8613437 ESRQRKGRTLTEAKKLMRERN-YFAAMMVNVGEADALITGYSRPYPTVIRPILESIQKDS
GOS_26940 SLLARRGVSPDLAKSIMRTNTTAIGAVMVHRGEADSLICGAVGEFRWHLNYIEQILGSK-
gi|8870682 QLMSRRGVTPDLAKVIMRTNTTAIAAIMVYCGDADSMVCGSFGQYLWHLNYVRQILAYD-
gi|2066869 KVMQRRGVTPDLAKAIMRTNTTAIGAIMVHRGEADSLLCGTFGEYRWHLNYVQQVLGGG-
Spomeroyi QLMERRGVTPDIAKAIMRTNTTAIGAIMVHRGEADSLICGTFGEYRWHLNYVEQVLGSK-
gi|1584252 EIAGRRGVTPDAARTLVRTSSTVISALAVKKGEADAMLCGIEGRFSRHLRHVRDIIGLAP
gi|1529713 QLMKRRGITQEQAQRAVISNTTVIGAIMVHRGEADAMICGTIGEYHDHYRVVQPLFGYRD
gi|7680888 KMMARKGISEQLARVEMRRRTTLIGSMLVKKGEADGMICGTISTTHRHLHFIDQVIGKRA
gi|1879253 KMMSRKGISAQMAKLEMRRRTTLIGAMLVEKGEADGMICGTVSTTHRHLHFIDQVIGKKE
. *.* : *. : . :.:: * *:**.:: * .: :

gi|8613437 GISKVAACNLMLTKQGPMFLADTTINLNPTAKDLVKISQMTSNLVKMFGMKPNVAMLSFS
GOS_26940 TLSPSGALSLMILEDGPLFIADTHVWADPTPMQIAQTAKGAARHVRRFGIEPQVALCSQS
gi|8870682 GAHPRGALSLMITEDEPLFIADTHVHPEPTPEQIADTVMAAANHVRRFGMKPNIALCSHS
gi|2066869 TYSPHGALSMMILEDGPLFIADTHVHVEPTPEQIAETVIGAARHVRRFGLAPKIALCSQS
Spomeroyi DLRPHGALSLMILEDGPLFIADTHVRSRPSPEELAEITLGAARHVRRFGIEPQIALCSQS
gi|1584252 GVRELAALSLLITPKGNLFLCDTQVQTEPNAADLAEMTILAAAHVRRFGIEPKVALLSHS
gi|1529713 GVSTAGAMNALLLPSGNTFIADTYVNHDPSPEELAEITLMAAESVRRFGIEPRVALLSHS
gi|7680888 GCSVYGAMNALVLPGRQIFLVDTHVNVDPTPAQLAEITIMAAEEVRRFGIEPKVALLSHS
gi|1879253 GAKVYAAMNALVLPNRQIFLVDTHVNVDPTPEQLAEITIMAAEEVRRFGIEPKIALLSHS
.* . :: *: ** :. *.. ::.. :: *. **: *.:*: * *

gi|8613437 NFGSTKNESSQKIREAVSYIHRNFPNAVVDGEIQADFALNPEMLAKEFPFSKLNGKKVNV
GOS_26940 QFGNLNSETGKKMRQALDILDTEKVTFTYEGEMNIDTALDPELRARLLPENR--------
gi|8870682 QFGNLDIDSGRRVRQAMALLEAREPDFAYEGEMHIDSALDPDLRARIFPNSRLQG-PANV
gi|2066869 QFGNISCDTGSRLRAAIEILDDKRRDFVYEGEMNIDTALDPELRERIFPNSRLEG-AANV
Spomeroyi QFGNQAEGSGQRLRQAIEILDSRPRDFVYEGEMNLDSALDPELRQRIFPNSRLYG-AANV
gi|1584252 NFGSNDTVCARRVRAALDILKDRAPELEVDGEMQAELALLPDARERILPHSRLQG-VANV
gi|1529713 NFGSADCPSASKMRKTLELVKARAPELMIDGEMHGDAALVESIRNDRMPDSPLKG-AANI
gi|7680888 NFGTSNAPSAQKMRDTLAILQERAPDLHVDGEMHGDVALDAALRKEILPESTLEG-EANL
gi|1879253 NFGTSNAPTAQKMRDTLAILRERAPDLQVDGEMHGDIALDANLRREVMPDSTLEG-DANL

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:**. . .:* :: : :**:: : ** :* .

gi|8613437 LIFPNLESANITYKLLKEMQG-AESIGPVILGLSKAVHIVQLGASVDEMVNMAALACVDA
GOS_26940 ------------------------------------------------------------
gi|8870682 LVFAYGDAASGVRNILKMRGG-ALEVGPILMGMGNRAHIVTPSITARGLLNISALAGTDV
gi|2066869 LIFAHADAASGVRNILKMRAG-GLEVGPILMGMGNRAHIVSPSITARGLLNMAAIAGTPV
Spomeroyi LIFAHADAASGVRNVLKMKAN-GIEVGPILMGMGNRAHIVTPSITARGLLNMAAIAGTPV
gi|1584252 LVMPDLDAADIAYNMIKVLGD-ALPVGPILMGTAKPAHILGPTVTARGIVNMTAVAVVEA
gi|1529713 LVMPNMEAARISYNLLRVSSSEGVTVGPVLMGVAKPVHILTPIASVRRIVNMVALAVVEA
gi|7680888 LVLPNIDAANIAYNLLKTAAGNNIAIGPILLGAAQPVHVLTESATVRRIVNMTALLVADV
gi|1879253 LVLPNIDAANISYNLLKTAAGNNIAIGPMLLGAAKPVHVLTASATVRRIVNMTALLVADV

gi|8613437 QQREKK
GOS_26940 ------
gi|8870682 THYS--
gi|2066869 AHYG--
Spomeroyi AHYG--
gi|1584252 QSEA--
gi|1529713 QTEPL-
gi|7680888 NAVR--
gi|1879253 IAAR--

Alignment curation form

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Curated alignment check

The GBLOCKS curated multiple sequence alignment (paste in Annotathon multiple alignment field):

Gblocks 0.91b Results

Processed file: input.fasta

Number of sequences: 9
Alignment assumed to be: Protein
New number of positions: 288 (selected positions are underlined in blue)

10 20 30 40 50 60
=========+=========+=========+=========+=========+=========+
gi|86134375|ref ------------------MSNSRKRHEALLYHAKPKPGKIAVVPTKKYATQHDLALAYSP
GOS_26940_Trans ------------------------------------------------------------
gi|88706826|ref --------------MDDDKSRQAARDAALRYHAYPKPGKLEIRATKPLANGQDLARAYSP
gi|206686971|gb -----------------MSDSQNLRQAALNYHEFPRPGKLEIRATKPMANGRDLARAYSP
Spomeroyi_gi|56 -----------------MSDQPSLRQAALDYHAFPKPGKLEIRATKPMANGRDLARAYSP
gi|158425280|re ----------------MSNISEDLKSGALVYHRSPKPGKLEIQATKPLGNQRDLALAYSP
gi|152971328|re -------------------MDEQLKQSALDFHEFPVPGKIQVSPTKPLATQRDLALAYSP
gi|76808889|ref ----------MSTSSSSSSSKEKLREAALDYHEFPTPGKVAIAPTKQMINQRDLALAYSP
gi|187925371|re MPSNVYSNPPSEARLMSTPVNSKLREAALDYHEFPTPGKIAIAPTKQMINQRDLALAYSP

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70 80 90 100 110 120

=========+=========+=========+=========+=========+=========+
gi|86134375|ref GVAEPCLEIAKDKNNIYKYTSKGNLVAVISNGTAVLGLGDIGPEASKPVMEGKGLLFKIF
GOS_26940_Trans ------------------------------------------------------------
gi|88706826|ref GVAEACLEIVKDPATAADYTARGNLVAVISNGSAVLGLGNIGGLAAKPVMEGKAVLFKNF
gi|206686971|gb GVAEACTEIQADAANAARYTSRGNLVAVVSNGSAVLGLGNIGALASKPVMEGKAVLFKNF
Spomeroyi_gi|56 GVAEACLEIKDNAAHAETYTARGNLVAVVSNGTAVLGLGNIGALASKPVMEGKAVLFKKF
gi|158425280|re GVAAACEAIKADPLQAAELTTRANLVAVVSNGTAVLGLGNIGPLASKPVMEGKAVLFKKF
gi|152971328|re GVAAPCLEIEKDPLAAYKYTARGNLVAVVSNGTAVLGLGNIGALAGKPVMEGKGVLFKKF
gi|76808889|ref GVAFACEEIVENPLNAARFTARSNLVGVVTNGTAVLGLGNIGPLASKPVMEGKAVLFKKF
gi|187925371|re GVAFACEEIVENPLNAARFTARSNLVGVVTNGTAVLGLGNIGPLASKPVMEGKAVLFKKF

370 380 390 400 410 420

=========+=========+=========+=========+=========+=========+
gi|86134375|ref AMKLAAVHALADLAKKSVPEQVNIVYDEVSLNFGKEYIIPKPFDPRLIYEIPPAVAKAAM
GOS_26940_Trans -----------------------------------------PFDPRLSSVVSSAVAEAAM
gi|88706826|ref AMQLACIDGIAALSRATTSAEAAEAYRGEQLVFGVDYLIPKPFDPRLMGVVASAVASAAM
gi|206686971|gb EMQIACVDGIAELARATTSAEAAAAYKGEQLNFGADYLIPKPFDPRLVAVVSSAVAKAAM
Spomeroyi_gi|56 AMQIACVEGIAELARITTSAEAAAAYQGEQLTFGADYLIPKPFDPRLVGVVSSAVARAAM
gi|158425280|re EMKMAAVEAIAALARETPSDVVARAYGGETRAFGADSIIPSPFDPRLILRIAPAVAKAAM
gi|152971328|re EMKLAAVHAIAELAHAEQSEVVASAYGDQDLSFGPEYIIPKPFDPRLIVKIAPAVAKAAM
gi|76808889|ref EMEIAAVNAIAELAQQEQSDIVATAYGIQDLSFGPEYLIPKPFDPRLIVKIAPAVAQAAM
gi|187925371|re EMEIAAVNAIAELARQEQSDIVATAYGIQDLSFGPEYLIPKPFDPRLIVKVAPAVAKAAM
###################

430 440 450 460 470 480

=========+=========+=========+=========+=========+=========+
gi|86134375|ref ESGVALEPISDWDAYREELMERSGSGSKEIRQIHNRAK---RNKKRIVFAEADHLDVLKA
GOS_26940_Trans QSGVATQPIKDIDAYRDALKQTVVKSAFLMRPVFEAAS---SSARRIVFAEGEDERVLRA
gi|88706826|ref ETGVATRPVEDLVAYRERLDASVFRSSMIMRPVFAAAA---LSQRRIVFAEGEDERVLRT
gi|206686971|gb ESGVATRPIEDITAYKQKLNQTVFKSALLMRPVFEAAR---AAARRIVFAEGEDERVLRA
Spomeroyi_gi|56 ESGVARRPITDLEAYRQKLNQSVFKSALLMRPVFEAAA---KAARRLVFAEGEDERVLRA
gi|158425280|re DTGVATRPIADFDAYNEKLDEFVFRSGFIMRPLFQRAK---QDKKRVIYAEGEDERVLRA
gi|152971328|re DSGVATRPIADFDAYIEKLSEFVYKTNLFMKPIFSQAR---KEPKRVVLAEGEETRVLHA
gi|76808889|ref DGGVATRPIEDMEAYKVHLQQFVYHSGTTMKPVFQIARGAPAEKKRVVFAEGEEERVLRA
gi|187925371|re DSGVAERPIEDMEAYEQHLQQFVYHSGTTMKPIFQLARGVEPEKKRIVFAEGEEERVLRA
##################################### ################

490 500 510 520 530 540

=========+=========+=========+=========+=========+=========+
gi|86134375|ref AQRVQEEKLGLPILLGRKEVILELKEEIGFT----EDVPIFDPKTDEEKERRDRFGIAYW
GOS_26940_Trans AQAVLEETSEVPIVIGRPEVIQQRCERLGLDIRPDRDFNIVNPQQD---DRYRDYWTSYH
gi|88706826|ref AQVIVEEMTDRPILIGRPEIIARRCEKAGLTIKPGEDFEVVNPEDD---SRHRRYWEAYL
gi|206686971|gb AQAILEETTETPILIGRPEVIERRCEKLGLDVRPGRDFQLVNPEND---PRYYDYWNSYH
Spomeroyi_gi|56 AQAILEETTETPILIGRPEVIEARCEKMGLSVRPGQDFQIVNPEND---PRYYDYWTSYH
gi|158425280|re AQAVIEEGIAHPILVARPSVLEARLQRFGLSIRPGKDFEVINPEDD---PRYRDFVRSYI
gi|152971328|re TQELVSLGLAKPILVGRPSVIEMRIQKLGLQIKAGVDFEIVNNESD---PRFKEYWSEYY
gi|76808889|ref VQIVVDEKLAKPILIGRPAVIEHRIQRYGLRLTPGVDFTIVNTEHD---ERYRDFWQTYF
gi|187925371|re MQIIVDEKLAKPILIGRPAVIEQRIARYGLRLIAGQDYTVVNTDHD---ERYRDFWQEYH
############################## ########## ##########

550 560 570 580 590 600

=========+=========+=========+=========+=========+=========+
gi|86134375|ref ESRQRKGRTLTEAKKLMRERN-YFAAMMVNVGEADALITGYSRPYPTVIRPILESIQKDS
GOS_26940_Trans SLLARRGVSPDLAKSIMRTNTTAIGAVMVHRGEADSLICGAVGEFRWHLNYIEQILGSK-
gi|88706826|ref QLMSRRGVTPDLAKVIMRTNTTAIAAIMVYCGDADSMVCGSFGQYLWHLNYVRQILAYD-
gi|206686971|gb KVMQRRGVTPDLAKAIMRTNTTAIGAIMVHRGEADSLLCGTFGEYRWHLNYVQQVLGGG-
Spomeroyi_gi|56 QLMERRGVTPDIAKAIMRTNTTAIGAIMVHRGEADSLICGTFGEYRWHLNYVEQVLGSK-
gi|158425280|re EIAGRRGVTPDAARTLVRTSSTVISALAVKKGEADAMLCGIEGRFSRHLRHVRDIIGLAP
gi|152971328|re QLMKRRGITQEQAQRAVISNTTVIGAIMVHRGEADAMICGTIGEYHDHYRVVQPLFGYRD
gi|76808889|ref KMMARKGISEQLARVEMRRRTTLIGSMLVKKGEADGMICGTISTTHRHLHFIDQVIGKRA
gi|187925371|re KMMSRKGISAQMAKLEMRRRTTLIGAMLVEKGEADGMICGTVSTTHRHLHFIDQVIGKKE
##################### ##################################

610 620 630 640 650 660

=========+=========+=========+=========+=========+=========+
gi|86134375|ref GISKVAACNLMLTKQGPMFLADTTINLNPTAKDLVKISQMTSNLVKMFGMKPNVAMLSFS
GOS_26940_Trans TLSPSGALSLMILEDGPLFIADTHVWADPTPMQIAQTAKGAARHVRRFGIEPQVALCSQS
gi|88706826|ref GAHPRGALSLMITEDEPLFIADTHVHPEPTPEQIADTVMAAANHVRRFGMKPNIALCSHS
gi|206686971|gb TYSPHGALSMMILEDGPLFIADTHVHVEPTPEQIAETVIGAARHVRRFGLAPKIALCSQS

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Spomeroyi_gi|56 DLRPHGALSLMILEDGPLFIADTHVRSRPSPEELAEITLGAARHVRRFGIEPQIALCSQS
gi|158425280|re GVRELAALSLLITPKGNLFLCDTQVQTEPNAADLAEMTILAAAHVRRFGIEPKVALLSHS
gi|152971328|re GVSTAGAMNALLLPSGNTFIADTYVNHDPSPEELAEITLMAAESVRRFGIEPRVALLSHS
gi|76808889|ref GCSVYGAMNALVLPGRQIFLVDTHVNVDPTPAQLAEITIMAAEEVRRFGIEPKVALLSHS
gi|187925371|re GAKVYAAMNALVLPNRQIFLVDTHVNVDPTPEQLAEITIMAAEEVRRFGIEPKIALLSHS
############################################################

670 680 690 700 710 720

=========+=========+=========+=========+=========+=========+
gi|86134375|ref NFGSTKNESSQKIREAVSYIHRNFPNAVVDGEIQADFALNPEMLAKEFPFSKLNGKKVNV
GOS_26940_Trans QFGNLNSETGKKMRQALDILDTEKVTFTYEGEMNIDTALDPELRARLLPENR--------
gi|88706826|ref QFGNLDIDSGRRVRQAMALLEAREPDFAYEGEMHIDSALDPDLRARIFPNSRLQG-PANV
gi|206686971|gb QFGNISCDTGSRLRAAIEILDDKRRDFVYEGEMNIDTALDPELRERIFPNSRLEG-AANV
Spomeroyi_gi|56 QFGNQAEGSGQRLRQAIEILDSRPRDFVYEGEMNLDSALDPELRQRIFPNSRLYG-AANV
gi|158425280|re NFGSNDTVCARRVRAALDILKDRAPELEVDGEMQAELALLPDARERILPHSRLQG-VANV
gi|152971328|re NFGSADCPSASKMRKTLELVKARAPELMIDGEMHGDAALVESIRNDRMPDSPLKG-AANI
gi|76808889|ref NFGTSNAPSAQKMRDTLAILQERAPDLHVDGEMHGDVALDAALRKEILPESTLEG-EANL
gi|187925371|re NFGTSNAPTAQKMRDTLAILRERAPDLQVDGEMHGDIALDANLRREVMPDSTLEG-DANL
###################################################

730 740 750 760 770 780

=========+=========+=========+=========+=========+=========+
gi|86134375|ref LIFPNLESANITYKLLKEMQG-AESIGPVILGLSKAVHIVQLGASVDEMVNMAALACVDA
GOS_26940_Trans ------------------------------------------------------------
gi|88706826|ref LVFAYGDAASGVRNILKMRGG-ALEVGPILMGMGNRAHIVTPSITARGLLNISALAGTDV
gi|206686971|gb LIFAHADAASGVRNILKMRAG-GLEVGPILMGMGNRAHIVSPSITARGLLNMAAIAGTPV
Spomeroyi_gi|56 LIFAHADAASGVRNVLKMKAN-GIEVGPILMGMGNRAHIVTPSITARGLLNMAAIAGTPV
gi|158425280|re LVMPDLDAADIAYNMIKVLGD-ALPVGPILMGTAKPAHILGPTVTARGIVNMTAVAVVEA
gi|152971328|re LVMPNMEAARISYNLLRVSSSEGVTVGPVLMGVAKPVHILTPIASVRRIVNMVALAVVEA
gi|76808889|ref LVLPNIDAANIAYNLLKTAAGNNIAIGPILLGAAQPVHVLTESATVRRIVNMTALLVADV
gi|187925371|re LVLPNIDAANISYNLLKTAAGNNIAIGPMLLGAAKPVHVLTASATVRRIVNMTALLVADV

======
gi|86134375|ref QQREKK
GOS_26940_Trans ------
gi|88706826|ref THYS--
gi|206686971|gb AHYG--
Spomeroyi_gi|56 AHYG--
gi|158425280|re QSEA--
gi|152971328|re QTEPL-
gi|76808889|ref NAVR--
gi|187925371|re IAAR--

Parameters used
Minimum Number Of Sequences For A Conserved Position: 5
Minimum Number Of Sequences For A Flanking Position: 8
Maximum Number Of Contiguous Nonconserved Positions: 8
Minimum Length Of A Block: 10
Allowed Gap Positions: None
Use Similarity Matrices: Yes

Flank positions of the 6 selected block(s)

Flanks: [402 457] [465 510] [517 526] [531 561] [564 597] [601 711]

New number of positions in input.fasta-gb: 288 (36% of the original 786 positions)

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Tree inference method form

Tree rendering form

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Infered tree

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Tree leaf renaming

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Tree rerooting and textual tree export

The phylogenetic tree in "text" format to be copied in the Annotathon "Tree" section (remember to add the taxonomic group definitions):

-------0.2-----

+------------------Congregibacter_litoralis_KT71_gi_88706826 [Add taxonomi

|
| +------Rhodobacterales_bacterium_Y4I_gi_206686971 [Add taxonomi
+---------------------+ |
| | ++
| +------++--------Silicibacter_pomeroyi_DSS-3_gi_56697770 [Add taxonomi
| |
| +-----------------GOS_26940_Translation_11-922_indirect_strand
|
+------------------+ +-----Burkholderia_pseudomallei_1710b_gi_76808889 [Add taxonomi
| | |
| | +------------------+
| | +---------+ +------Burkholderia_phytofirmans_PsJN_gi_187925371 [Add taxonomi
| | | |
| | | +------------------------Klebsiella_pneumoniae_subsp._pneumoniae_gi_152971328 [Add taxonomi
| +--+
| |
| +----------------------------Azorhizobium_caulinodans_ORS_571_gi_158425280 [Add taxonomi
|
+-----------------------------------------------------------------Polaribacter_dokdonensis_MED152_gi_86134375 [Add taxonomi

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This page was last modified on 17 December 2008, at 17:25.

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