Provided by: libbio-perl-perl_1.7.8-1_all 
NAME
Bio::SeqFeature::Tools::Unflattener - turns flat list of genbank-sourced features into a
nested SeqFeatureI hierarchy
SYNOPSIS
# standard / generic use - unflatten a genbank record
use Bio::SeqIO;
use Bio::SeqFeature::Tools::Unflattener;
# generate an Unflattener object
$unflattener = Bio::SeqFeature::Tools::Unflattener->new;
# first fetch a genbank SeqI object
$seqio =
Bio::SeqIO->new(-file=>'AE003644.gbk',
-format=>'GenBank');
my $out =
Bio::SeqIO->new(-format=>'asciitree');
while ($seq = $seqio->next_seq()) {
# get top level unflattended SeqFeatureI objects
$unflattener->unflatten_seq(-seq=>$seq,
-use_magic=>1);
$out->write_seq($seq);
@top_sfs = $seq->get_SeqFeatures;
foreach my $sf (@top_sfs) {
# do something with top-level features (eg genes)
}
}
DESCRIPTION
Most GenBank entries for annotated genomic DNA contain a flat list of features. These
features can be parsed into an equivalent flat list of Bio::SeqFeatureI objects using the
standard Bio::SeqIO classes. However, it is often desirable to unflatten this list into
something resembling actual gene models, in which genes, mRNAs and CDSs are nested
according to the nature of the gene model.
The BioPerl object model allows us to store these kind of associations between SeqFeatures
in containment hierarchies -- any SeqFeatureI object can contain nested SeqFeatureI
objects. The Bio::SeqFeature::Tools::Unflattener object facilitates construction of these
hierarchies from the underlying GenBank flat-feature-list representation.
For example, if you were to look at a typical GenBank DNA entry, say, AE003644, you would
see a flat list of features:
source
gene CG4491
mRNA CG4491-RA
CDS CG4491-PA
gene tRNA-Pro
tRNA tRNA-Pro
gene CG32954
mRNA CG32954-RA
mRNA CG32954-RC
mRNA CG32954-RB
CDS CG32954-PA
CDS CG32954-PB
CDS CG32954-PC
These features have sequence locations, but it is not immediately clear how to write code
such that each mRNA is linked to the appropriate CDS (other than relying on IDs which is
very bad)
We would like to convert the above list into the containment hierarchy, shown below:
source
gene
mRNA CG4491-RA
CDS CG4491-PA
exon
exon
gene
tRNA tRNA-Pro
exon
gene
mRNA CG32954-RA
CDS CG32954-PA
exon
exon
mRNA CG32954-RC
CDS CG32954-PC
exon
exon
mRNA CG32954-RB
CDS CG32954-PB
exon
exon
Where each feature is nested underneath its container. Note that exons have been
automatically inferred (even for tRNA genes).
We do this using a call on a Bio::SeqFeature::Tools::Unflattener object
@sfs = $unflattener->unflatten_seq(-seq=>$seq);
This would return a list of the top level (i.e. container) SeqFeatureI objects - in this
case, genes. Other top level features are possible; for instance, the source feature which
is always present, and other features such as variation or misc_feature types.
The containment hierarchy can be accessed using the get_SeqFeature() call on any feature
object - see Bio::SeqFeature::FeatureHolderI. The following code will traverse the
containment hierarchy for a feature:
sub traverse {
$sf = shift; # $sf isa Bio::SeqfeatureI
# ...do something with $sf!
# depth first traversal of containment tree
@contained_sfs = $sf->get_SeqFeatures;
traverse($_) foreach @contained_sfs;
}
Once you have built the hierarchy, you can do neat stuff like turn the features into
'rich' feature objects (eg Bio::SeqFeature::Gene::GeneStructure) or convert to a suitable
format such as GFF3 or chadoxml (after mapping to the Sequence Ontology); this step is not
described here.
USING MAGIC
Due to the quixotic nature of how features are stored in GenBank/EMBL/DDBJ, there is no
guarantee that the default behaviour of this module will produce perfect results.
Sometimes it is hard or impossible to build a correct containment hierarchy if the
information provided is simply too lossy, as is often the case. If you care deeply about
your data, you should always manually inspect the resulting containment hierarchy; you may
have to customise the algorithm for building the hierarchy, or even manually tweak the
resulting hierarchy. This is explained in more detail further on in the document.
However, if you are satisfied with the default behaviour, then you do not need to read any
further. Just make sure you set the parameter use_magic - this will invoke incantations
which will magically produce good results no matter what the idiosyncracies of the
particular GenBank record in question.
For example
$unflattener->unflatten_seq(-seq=>$seq,
-use_magic=>1);
The success of this depends on the phase of the moon at the time the entry was submitted
to GenBank. Note that the magical recipe is being constantly improved, so the results of
invoking magic may vary depending on the bioperl release.
If you are skeptical of magic, or you wish to exact fine grained control over how the
entry is unflattened, or you simply wish to understand more about how this crazy stuff
works, then read on!
PROBLEMATIC DATA AND INCONSISTENCIES
Occasionally the Unflattener will have problems with certain records. For example, the
record may contain inconsistent data - maybe there is an exon entry that has no
corresponding mRNA location.
The default behaviour is to throw an exception reporting the problem, if the problem is
relatively serious - for example, inconsistent data.
You can exert more fine grained control over this - perhaps you want the Unflattener to do
the best it can, and report any problems. This can be done - refer to the methods.
error_threshold()
get_problems()
report_problems()
ignore_problems()
ALGORITHM
This is the default algorithm; you should be able to override any part of it to customise.
The core of the algorithm is in two parts
Partitioning the flat feature list into groups
Resolving the feature containment hierarchy for each group
There are other optional steps after the completion of these two steps, such as inferring
exons; we now describe in more detail what is going on.
Partitioning into groups
First of all the flat feature list is partitioned into groups.
The default way of doing this is to use the gene attribute; if we look at two features
from GenBank accession AE003644.3:
gene 20111..23268
/gene="noc"
/locus_tag="CG4491"
/note="last curated on Thu Dec 13 16:51:32 PST 2001"
/map="35B2-35B2"
/db_xref="FLYBASE:FBgn0005771"
mRNA join(20111..20584,20887..23268)
/gene="noc"
/locus_tag="CG4491"
/product="CG4491-RA"
/db_xref="FLYBASE:FBgn0005771"
Both these features share the same /gene tag which is "noc", so they correspond to the
same gene model (the CDS feature is not shown, but this also has a tag-value /gene="noc").
Not all groups need to correspond to gene models, but this is the most common use case;
later on we shall describe how to customise the grouping.
Sometimes other tags have to be used; for instance, if you look at the entire record for
AE003644.3 you will see you actually need the use the /locus_tag attribute. This attribute
is actually not present in most records!
You can override this:
$collection->unflatten_seq(-seq=>$seq, -group_tag=>'locus_tag');
Alternatively, if you -use_magic, the object will try and make a guess as to what the
correct group_tag should be.
At the end of this step, we should have a list of groups - there is no structure within a
group; the group just serves to partition the flat features. For the example data above,
we would have the following groups.
[ source ]
[ gene mRNA CDS ]
[ gene mRNA CDS ]
[ gene mRNA CDS ]
[ gene mRNA mRNA mRNA CDS CDS CDS ]
Multicopy Genes
Multicopy genes are usually rRNAs or tRNAs that are duplicated across the genome. Because
they are functionally equivalent, and usually have the same sequence, they usually have
the same group_tag (ie gene symbol); they often have a /note tag giving copy number. This
means they will end up in the same group. This is undesirable, because they are spatially
disconnected.
There is another step, which involves splitting spatially disconnected groups into
distinct groups
this would turn this
[gene-rrn3 rRNA-rrn3 gene-rrn3 rRNA-rrn3]
into this
[gene-rrn3 rRNA-rrn3] [gene-rrn3 rRNA-rrn3]
based on the coordinates
What next?
The next step is to add some structure to each group, by making containment hierarchies,
trees that represent how the features interrelate
Resolving the containment hierarchy
After the grouping is done, we end up with a list of groups which probably contain
features of type 'gene', 'mRNA', 'CDS' and so on.
Singleton groups (eg the 'source' feature) are ignored at this stage.
Each group is itself flat; we need to add an extra level of organisation. Usually this is
because different spliceforms (represented by the 'mRNA' feature) can give rise to
different protein products (indicated by the 'CDS' feature). We want to correctly
associate mRNAs to CDSs.
We want to go from a group like this:
[ gene mRNA mRNA mRNA CDS CDS CDS ]
to a containment hierarchy like this:
gene
mRNA
CDS
mRNA
CDS
mRNA
CDS
In which each CDS is nested underneath the correct corresponding mRNA.
For entries that contain no alternate splicing, this is simple; we know that the group
[ gene mRNA CDS ]
Must resolve to the tree
gene
mRNA
CDS
How can we do this in entries with alternate splicing? The bad news is that there is no
guaranteed way of doing this correctly for any GenBank entry. Occasionally the submission
will have been done in such a way as to reconstruct the containment hierarchy. However,
this is not consistent across databank entries, so no generic solution can be provided by
this object. This module does provide the framework within which you can customise a
solution for the particular dataset you are interested in - see later.
The good news is that there is an inference we can do that should produce pretty good
results the vast majority of the time. It uses splice coordinate data - this is the
default behaviour of this module, and is described in detail below.
Using splice site coordinates to infer containment
If an mRNA is to be the container for a CDS, then the splice site coordinates (or intron
coordinates, depending on how you look at it) of the CDS must fit inside the splice site
coordinates of the mRNA.
Ambiguities can still arise, but the results produced should still be reasonable and
consistent at the sequence level. Look at this fake example:
mRNA XXX---XX--XXXXXX--XXXX join(1..3,7..8,11..16,19..23)
mRNA XXX-------XXXXXX--XXXX join(1..3,11..16,19..23)
CDS XXXX--XX join(13..16,19..20)
CDS XXXX--XX join(13..16,19..20)
[obviously the positions have been scaled down]
We cannot unambiguously match mRNA with CDS based on splice sites, since both CDS share
the splice site locations 16^17 and 18^19. However, the consequences of making a wrong
match are probably not very severe. Any annotation data attached to the first CDS is
probably identical to the seconds CDS, other than identifiers.
The default behaviour of this module is to make an arbitrary call where it is ambiguous
(the mapping will always be bijective; i.e. one mRNA -> one CDS).
[TODO: NOTE: not tested on EMBL data, which may not be bijective; ie two mRNAs can share
the same CDS??]
This completes the building of the containment hierarchy; other optional step follow
POST-GROUPING STEPS
Inferring exons from mRNAs
This step always occurs if -use_magic is invoked.
In a typical GenBank entry, the exons are implicit. That is they can be inferred from the
mRNA location.
For example:
mRNA join(20111..20584,20887..23268)
This tells us that this particular transcript has two exons. In bioperl, the mRNA feature
will have a 'split location'.
If we call
$unflattener->feature_from_splitloc(-seq=>$seq);
This will generate the necessary exon features, and nest them under the appropriate mRNAs.
Note that the mRNAs will no longer have split locations - they will have simple locations
spanning the extent of the exons. This is intentional, to avoid redundancy.
Occasionally a GenBank entry will have both implicit exons (from the mRNA location) and
explicit exon features.
In this case, exons will still be transferred. Tag-value data from the explicit exon will
be transferred to the implicit exon. If exons are shared between mRNAs these will be
represented by different objects. Any inconsistencies between implicit and explicit will
be reported.
tRNAs and other noncoding RNAs
exons will also be generated from these features
Inferring mRNAs from CDS
Some GenBank entries represent gene models using features of type gene, mRNA and CDS; some
entries just use gene and CDS.
If we only have gene and CDS, then the containment hierarchies will look like this:
gene
CDS
If we want the containment hierarchies to be uniform, like this
gene
mRNA
CDS
Then we must create an mRNA feature. This will have identical coordinates to the CDS. The
assumption is that there is either no untranslated region, or it is unknown.
To do this, we can call
$unflattener->infer_mRNA_from_CDS(-seq=>$seq);
This is taken care of automatically, if -use_magic is invoked.
ADVANCED
Customising the grouping of features
The default behaviour is suited mostly to building models of protein coding genes and
noncoding genes from genbank genomic DNA submissions.
You can change the tag used to partition the feature by passing in a different group_tag
argument - see the unflatten_seq() method
Other behaviour may be desirable. For example, even though SNPs (features of type
'variation' in GenBank) are not actually part of the gene model, it may be desirable to
group SNPs that overlap or are nearby gene models.
It should certainly be possible to extend this module to do this. However, I have yet to
code this part!!! If anyone would find this useful let me know.
In the meantime, you could write your own grouping subroutine, and feed the results into
unflatten_groups() [see the method documentation below]
Customising the resolution of the containment hierarchy
Once the flat list of features has been partitioned into groups, the method
unflatten_group() is called on each group to build a tree.
The algorithm for doing this is described above; ambiguities are resolved by using splice
coordinates. As discussed, this can be ambiguous.
Some submissions may contain information in tags/attributes that hint as to the mapping
that needs to be made between the features.
For example, with the Drosophila Melanogaster release 3 submission, we see that CDS
features in alternately spliced mRNAs have a form like this:
CDS join(145588..145686,145752..146156,146227..146493)
/locus_tag="CG32954"
/note="CG32954 gene product from transcript CG32954-RA"
^^^^^^^^^^^^^^^^^^^^^^^^^^^
/codon_start=1
/product="CG32954-PA"
/protein_id="AAF53403.1"
/db_xref="GI:7298167"
/db_xref="FLYBASE:FBgn0052954"
/translation="MSFTLTNKNVIFVAGLGGIGLDTSKELLKRDLKNLVILDRIENP..."
Here the /note tag provides the clue we need to link CDS to mRNA (highlighted with ^^^^).
We just need to find the mRNA with the tag
/product="CG32954-RA"
I have no idea how consistent this practice is across submissions; it is consistent for
the fruitfly genome submission.
We can customise the behaviour of unflatten_group() by providing our own resolver method.
This obviously requires a bit of extra programming, but there is no way to get around
this.
Here is an example of how to pass in your own resolver; this example basically checks the
parent (container) /product tag to see if it matches the required string in the child
(contained) /note tag.
$unflattener->unflatten_seq(-seq=>$seq,
-group_tag=>'locus_tag',
-resolver_method=>sub {
my $self = shift;
my ($sf, @candidate_container_sfs) = @_;
if ($sf->has_tag('note')) {
my @notes = $sf->get_tag_values('note');
my @trnames = map {/from transcript\s+(.*)/;
$1} @notes;
@trnames = grep {$_} @trnames;
my $trname;
if (@trnames == 0) {
$self->throw("UNRESOLVABLE");
}
elsif (@trnames == 1) {
$trname = $trnames[0];
}
else {
$self->throw("AMBIGUOUS: @trnames");
}
my @container_sfs =
grep {
my ($product) =
$_->has_tag('product') ?
$_->get_tag_values('product') :
('');
$product eq $trname;
} @candidate_container_sfs;
if (@container_sfs == 0) {
$self->throw("UNRESOLVABLE");
}
elsif (@container_sfs == 1) {
# we got it!
return $container_sfs[0];
}
else {
$self->throw("AMBIGUOUS");
}
}
});
the resolver method is only called when there is more than one spliceform.
Parsing mRNA records
Some of the entries in sequence databanks are for mRNA sequences as well as genomic DNA.
We may want to build models from these too.
NOT YET DONE - IN PROGRESS!!!
Open question - what would these look like?
Ideally we would like a way of combining a mRNA record with the corresponding SeFeature
entry from the appropriate genomic DNA record. This could be problemmatic in some cases -
for example, the mRNA sequences may not match 100% (due to differences in strain, assembly
problems, sequencing problems, etc). What then...?
SEE ALSO
Feature table description
http://www.ebi.ac.uk/embl/Documentation/FT_definitions/feature_table.html
FEEDBACK
Mailing Lists
User feedback is an integral part of the evolution of this and other Bioperl modules. Send
your comments and suggestions preferably to the Bioperl mailing lists Your participation
is much appreciated.
bioperl-l@bioperl.org - General discussion
http://bioperl.org/wiki/Mailing_lists - About the mailing lists
Support
Please direct usage questions or support issues to the mailing list:
bioperl-l@bioperl.org
rather than to the module maintainer directly. Many experienced and reponsive experts will
be able look at the problem and quickly address it. Please include a thorough description
of the problem with code and data examples if at all possible.
Reporting Bugs
report bugs to the Bioperl bug tracking system to help us keep track the bugs and their
resolution. Bug reports can be submitted via the web:
https://github.com/bioperl/bioperl-live/issues
AUTHOR - Chris Mungall
Email: cjm@fruitfly.org
APPENDIX
The rest of the documentation details each of the object methods. Internal methods are
usually preceded with a _
new
Title : new
Usage : $unflattener = Bio::SeqFeature::Tools::Unflattener->new();
$unflattener->unflatten_seq(-seq=>$seq);
Function: constructor
Example :
Returns : a new Bio::SeqFeature::Tools::Unflattener
Args : see below
Arguments
-seq : A L<Bio::SeqI> object (optional)
the sequence to unflatten; this can also be passed in
when we call unflatten_seq()
-group_tag : a string representing the /tag used to partition flat features
(see discussion above)
seq
Title : seq
Usage : $unflattener->seq($newval)
Function:
Example :
Returns : value of seq (a Bio::SeqI)
Args : on set, new value (a Bio::SeqI, optional)
The Bio::SeqI object should hold a flat list of Bio::SeqFeatureI objects; this is the list
that will be unflattened.
The sequence object can also be set when we call unflatten_seq()
group_tag
Title : group_tag
Usage : $unflattener->group_tag($newval)
Function:
Example :
Returns : value of group_tag (a scalar)
Args : on set, new value (a scalar or undef, optional)
This is the tag that will be used to collect elements from the flat feature list into
groups; for instance, if we look at two typical GenBank features:
gene 20111..23268
/gene="noc"
/locus_tag="CG4491"
/note="last curated on Thu Dec 13 16:51:32 PST 2001"
/map="35B2-35B2"
/db_xref="FLYBASE:FBgn0005771"
mRNA join(20111..20584,20887..23268)
/gene="noc"
/locus_tag="CG4491"
/product="CG4491-RA"
/db_xref="FLYBASE:FBgn0005771"
We can see that these comprise the same gene model because they share the same /gene
attribute; we want to collect these together in groups.
Setting group_tag is optional. The default is to use 'gene'. In the example above, we
could also use /locus_tag
partonomy
Title : partonomy
Usage : $unflattener->partonomy({mRNA=>'gene', CDS=>'mRNA')
Function:
Example :
Returns : value of partonomy (a scalar)
Args : on set, new value (a scalar or undef, optional)
A hash representing the containment structure that the seq_feature nesting should conform
to; each key represents the contained (child) type; each value represents the container
(parent) type.
structure_type
Title : structure_type
Usage : $unflattener->structure_type($newval)
Function:
Example :
Returns : value of structure_type (a scalar)
Args : on set, new value (an int or undef, optional)
GenBank entries conform to different flavours, or structure types. Some have mRNAs, some
do not.
Right now there are only two base structure types defined. If you set the structure type,
then appropriate unflattening action will be taken. The presence or absence of explicit
exons does not affect the structure type.
If you invoke -use_magic then this will be set automatically, based on the content of the
record.
Type 0 (DEFAULT)
typically contains
source
gene
mRNA
CDS
with this structure type, we want the seq_features to be nested like this
gene
mRNA
CDS
exon
exons and introns are implicit from the mRNA 'join' location
to get exons from the mRNAs, you will need this call (see below)
$unflattener->feature_from_splitloc(-seq=>$seq);
Type 1
typically contains
source
gene
CDS
exon [optional]
intron [optional]
there are no mRNA features
with this structure type, we want the seq_features to be nested like this
gene
CDS
exon
intron
exon and intron may or may not be present; they may be implicit from the CDS 'join'
location
get_problems
Title : get_problems
Usage : @probs = get_problems()
Function: Get the list of problem(s) for this object.
Example :
Returns : An array of [severity, description] pairs
Args :
In the course of unflattening a record, problems may occur. Some of these problems are
non-fatal, and can be ignored.
Problems are represented as arrayrefs containing a pair [severity, description]
severity is a number, the higher, the more severe the problem
the description is a text string
clear_problems
Title : clear_problems
Usage :
Function: resets the problem list to empty
Example :
Returns :
Args :
report_problems
Title : report_problems
Usage : $unflattener->report_problems(\*STDERR);
Function:
Example :
Returns :
Args : FileHandle (defaults to STDERR)
ignore_problems
Title : ignore_problems
Usage : $obj->ignore_problems();
Function:
Example :
Returns :
Args :
Unflattener is very particular about problems it finds along the way. If you have set the
error_threshold such that less severe problems do not cause exceptions, Unflattener still
expects you to report_problems() at the end, so that the user of the module is aware of
any inconsistencies or problems with the data. In fact, a warning will be produced if
there are unreported problems. To silence, this warning, call the ignore_problems() method
before the Unflattener object is destroyed.
error_threshold
Title : error_threshold
Usage : $obj->error_threshold($severity)
Function:
Example :
Returns : value of error_threshold (a scalar)
Args : on set, new value (an integer)
Sets the threshold above which errors cause this module to throw an exception. The default
is 0; all problems with a severity > 0 will cause an exception.
If you raise the threshold to 1, then the unflattening process will be more lax; problems
of severity==1 are generally non-fatal, but may indicate that the results should be
inspected, for example, to make sure there is no data loss.
unflatten_seq
Title : unflatten_seq
Usage : @sfs = $unflattener->unflatten_seq($seq);
Function: turns a flat list of features into a list of holder features
Example :
Returns : list of Bio::SeqFeatureI objects
Args : see below
partitions a list of features then arranges them in a nested tree; see above for full
explanation.
note - the Bio::SeqI object passed in will be modified
Arguments
-seq : a Bio::SeqI object; must contain Bio::SeqFeatureI objects
(this is optional if seq has already been set)
-use_magic: if TRUE (ie non-zero) then magic will be invoked;
see discussion above.
-resolver_method: a CODE reference
see the documentation above for an example of
a subroutine that can be used to resolve hierarchies
within groups.
this is optional - if nothing is supplied, a default
subroutine will be used (see below)
-group_tag: a string
[ see the group_tag() method ]
this overrides the default group_tag which is 'gene'
unflatten_groups
Title : unflatten_groups
Usage :
Function: iterates over groups, calling unflatten_group() [see below]
Example :
Returns : list of Bio::SeqFeatureI objects that are holders
Args : see below
Arguments
-groups: list of list references; inner list is of Bio::SeqFeatureI objects
e.g. ( [$sf1], [$sf2, $sf3, $sf4], [$sf5, ...], ...)
-resolver_method: a CODE reference
see the documentation above for an example of
a subroutine that can be used to resolve hierarchies
within groups.
this is optional - a default subroutine will be used
NOTE: You should not need to call this method, unless you want fine grained control over
how the unflattening process.
unflatten_group
Title : unflatten_group
Usage :
Function: nests a group of features into a feature containment hierarchy
Example :
Returns : Bio::SeqFeatureI objects that holds other features
Args : see below
Arguments
-group: reference to list of Bio::SeqFeatureI objects
-resolver_method: a CODE reference
see the documentation above for an example of
a subroutine that can be used to resolve hierarchies
within groups
this is optional - a default subroutine will be used
NOTE: You should not need to call this method, unless you want fine grained control over
how the unflattening process.
feature_from_splitloc
Title : feature_from_splitloc
Usage : $unflattener->feature_from_splitloc(-features=>$sfs);
Function:
Example :
Returns :
Args : see below
At this time all this method does is generate exons for mRNA or other RNA features
Arguments:
-feature: a Bio::SeqFeatureI object (that conforms to Bio::FeatureHolderI)
-seq: a Bio::SeqI object that contains Bio::SeqFeatureI objects
-features: an arrayref of Bio::SeqFeatureI object
infer_mRNA_from_CDS
Title : infer_mRNA_from_CDS
Usage :
Function:
Example :
Returns :
Args :
given a "type 1" containment hierarchy
gene
CDS
exon
this will infer the uniform "type 0" containment hierarchy
gene
mRNA
CDS
exon
all the children of the CDS will be moved to the mRNA
a "type 2" containment hierarchy is mixed type "0" and "1" (for example, see
ftp.ncbi.nih.gov/genomes/Schizosaccharomyces_pombe/)
remove_types
Title : remove_types
Usage : $unf->remove_types(-seq=>$seq, -types=>["mRNA"]);
Function:
Example :
Returns :
Args :
removes features of a set type
useful for pre-filtering a genbank record; eg to get rid of STSs
also, there is no way to unflatten ftp.ncbi.nih.gov/genomes/Schizosaccharomyces_pombe/
UNLESS the bogus mRNAs in these records are removed (or changed to a different type) -
they just confuse things too much