[Bioperl-l] additional methods for Bio::SeqUtils for in-silico cloning

Frank Schwach fs5 at sanger.ac.uk
Wed Jan 11 13:16:53 EST 2012


Hi Roy and Chris,

I have made the changes to the code now. As you suggested, feature ends 
no longer change type and I insert a note instead to inform about the 
deletion (or insertion), showing the length and position.
I have also added a feature to annotate deletion sites themselves (with 
IN-BETWEEN locations).

Roy's test script now prints:

LOCUS       seq-accession_number            7 bp    dna     linear   UNK
ACCESSION   unknown
FEATURES             Location/Qualifiers
      CDS             join(2..3,4..6)
                      /note="3bp internal deletion between pos 3 and 4"
      CDS             2..3
                      /note="2bp deleted from feature end"
      misc_feature    3^4
                      /note="deletion of 3bp"
ORIGIN
         1 aaaaaaa
//


or, if you add strand information (-1 in this case) to the second feature:

LOCUS       seq-accession_number            7 bp    dna     linear   UNK
ACCESSION   unknown
FEATURES             Location/Qualifiers
      CDS             join(2..3,4..6)
                      /note="3bp internal deletion between pos 3 and 4"
      CDS             complement(2..3)
                      /note="2bp deleted from feature 5' end"
      misc_feature    3^4
                      /note="deletion of 3bp"
ORIGIN
         1 aaaaaaa
//

I have comitted this along with some bugfixes to my master branch on GitHub
https://github.com/fschwach/bioperl-live
so it's now also in my existing pull request.

I'm still wondering if cloning the sequence objects rather than calling 
'new' on their respective classes would be an option inside 'delete' and 
'insert'?
I'm experimenting with this for my own purposes because I have to work 
with custom sub-classes of Bio::Seq which have additional attributes and 
therefore set 'can_call_new' to false.
Without cloning the objects, I first have to convert the custom 
Bio::Seq::Foo objects to standard Bio::Seq, which I would like to avoid.
Is there any reason why something like Clone::Fast should not be used in 
this case? It seems to work for me but there may be situations where 
this is going to blow up which I am not aware of.
Cloning rather than calling new could be made an option in 
Bio::SeqUtils. I have most of the code for that already.

Frank











On 10/01/12 17:31, Roy Chaudhuri wrote:
> Or without the typo:
>
> CDS             join(2..3,4..6)
>                 /note="3 bp internal deletion"
> CDS             2..3
>                 /note="2 bp deleted from 3' end"
>
> On 10/01/2012 17:27, Roy Chaudhuri wrote:
>> I think it's me that didn't explain very well - I was talking about
>> overlapping (rather than spanning) a deletion, although I think the same
>> principle applies to the spanning example you gave. Here's some test 
>> code:
>>
>> #!/usr/bin/perl
>> use warnings FATAL=>qw(all);
>> use strict;
>> use Bio::Seq;
>> use Bio::SeqIO;
>> use Bio::SeqUtils;
>> use Bio::SeqFeature::Generic;
>> my $seq=Bio::Seq->new(-id=>'seq', -seq=>'AAAAAAAAAA');
>> $seq->add_SeqFeature(Bio::SeqFeature::Generic->new(-primary_tag=>'CDS',
>>                                                      -start=>2,
>>                                                      -end=>9));
>>
>> $seq->add_SeqFeature(Bio::SeqFeature::Generic->new(-primary_tag=>'CDS',
>>                                                      -start=>2,
>>                                                      -end=>5));
>> my $out=Bio::SeqIO->newFh(-format=>'genbank');
>> my $trunc=Bio::SeqUtils->delete($seq, 4, 6);
>> print $out $trunc;
>>
>>
>> This currently outputs:
>> LOCUS       seq-accession_number            7 bp    dna     linear   UNK
>> ACCESSION   unknown
>> FEATURES             Location/Qualifiers
>>        CDS             join(2..>3,<4..6)
>>        CDS             2..>3
>> ORIGIN
>>           1 aaaaaaa
>> //
>>
>> However, I was suggesting that the feature table should be something 
>> like:
>> CDS             join(2..3,4..6)
>>                   /note="3 bp internal deletion"
>> CDS             join(2..3)
>>                   /note="2 bp deleted from 3' end"
>>
>> Fuzzy locations are intended to represent features which have boundaries
>> spanning outside of the sequence. For a defined deletion that's not the
>> case, the boundaries of the feature aren't unknown, they have been
>> specifically altered.
>>
>> Hope this is clearer.
>> Cheers,
>> Roy.
>>
>> On 10/01/2012 16:47, Frank Schwach wrote:
>>> Hi Roy,
>>>
>>> Sorry, I hadn't explained that very well: it's not the outer boundaries
>>> of the feature that become fuzzy but the "inner" ones of the split
>>> locations:
>>>
>>>    --------------------           a feature's location
>>> ==========xxxx================= sequence
>>>
>>>
>>>    ---------                     sublocation 1
>>>             --------             sublocation 2
>>> ===============================
>>>
>>> x= sequence to delete
>>> The feature's location has changed from Simple to Split.
>>>
>>> Sublocation 1:
>>> start is still EXACT and has not changed
>>> end is now AFTER because this is not a true end of the feature
>>>
>>> Sublocation 2:
>>> start is BEFORE
>>> end is EXACT (but shifted)
>>>
>>> I hope this makes more sense(?)
>>>
>>> Cheers,
>>>
>>> Frank
>>>
>>>
>>>
>>> On Tue, 2012-01-10 at 15:25 +0000, Roy Chaudhuri wrote:
>>>> Hi Frank,
>>>>
>>>> Looks good to me. One thing I'm not sure about - why do features
>>>> overlapping a deletion become fuzzy? That behaviour is in
>>>> trunc_with_features because it's intended to represent a taking a
>>>> subregion of a larger sequence, but if you're representing an internal
>>>> deletion then the boundaries of the overlapping feature aren't 
>>>> unknown,
>>>> they have been specifically altered. Maybe you could give absolute
>>>> coordinates, but add a note indicating that the 5' or 3' end has been
>>>> truncated by however many bases.
>>>>
>>>> Cheers,
>>>> Roy.
>>>>
>>>> On 10/01/2012 13:10, Frank Schwach wrote:
>>>>> Hi Chris,
>>>>>
>>>>> I have made the changes in a Git fork and made the pull request now.
>>>>> If this is accepted into BioPerl I can also write a little SeqUtils
>>>>> HOWTO for the BioPerl wiki.
>>>>>
>>>>> Frank
>>>>>
>>>>>
>>>>> On Mon, 2012-01-09 at 18:29 +0000, Fields, Christopher J wrote:
>>>>>> Sounds very promising!  The easiest way to contribute is via a 
>>>>>> fork of the code on Github with a pull request (as you already 
>>>>>> know, being a contributor to the Primer3 modules).
>>>>>>
>>>>>> chris
>>>>>>
>>>>>> On Jan 9, 2012, at 11:10 AM, Frank Schwach wrote:
>>>>>>
>>>>>>> Hi all,
>>>>>>>
>>>>>>> I needed to manipulate Bio::Seq objects with annotations and 
>>>>>>> sequence
>>>>>>> features to simulate molecular cloning techniques, e.g. to cut a 
>>>>>>> vector
>>>>>>> and insert a fragment into it while preserving all the 
>>>>>>> annotations and
>>>>>>> moving the features accordingly.
>>>>>>> My main aim was to split features that span deletion/insertion 
>>>>>>> sites in
>>>>>>> a meaningful way, which can not be done with the currently availble
>>>>>>> methods.
>>>>>>> I have modified Bio::SeqUtils so that I have the following new 
>>>>>>> methods:
>>>>>>>
>>>>>>> delete
>>>>>>> ======
>>>>>>> removes a segment from a sequence object and adjusts positions 
>>>>>>> and types
>>>>>>> of locations of sequence features:
>>>>>>> - locations of features that span the deletion sites are turned 
>>>>>>> into
>>>>>>> Splits.
>>>>>>> - locations that extend into the deleted region are turned to 
>>>>>>> Fuzzy to
>>>>>>> indicate that their true start/end was lost.
>>>>>>> - locations contained inside the deleted regions are lost.
>>>>>>> - other features are shifted according to the length of the 
>>>>>>> deletion.
>>>>>>>
>>>>>>> insert
>>>>>>> ======
>>>>>>> adds a Bio::Seq object into another one between specified insertion
>>>>>>> sites. This also affects the features on the recipient sequence:
>>>>>>> - locations of features that span the insertion site are split but
>>>>>>> position types are not turned to Fuzzy because no part of the 
>>>>>>> original
>>>>>>> feature is lost.
>>>>>>> - other features are shifted according to the length of the 
>>>>>>> insertion.
>>>>>>>
>>>>>>> ligate
>>>>>>> ======
>>>>>>> just for convenience. Supply a recipient, a fragment and one or two
>>>>>>> sites to cut the recipient. Can also flip the fragment if required.
>>>>>>> Simply calls delete [, reverse_complement_with_features] and 
>>>>>>> insert in
>>>>>>> turn.
>>>>>>>
>>>>>>>
>>>>>>> One situation I haven't handled yet is a deletion that spans the 
>>>>>>> origin
>>>>>>> of a circular molecule but that should be a rare thing to do 
>>>>>>> anyway. The
>>>>>>> code currently throws an error if this is attempted.
>>>>>>>
>>>>>>> I'm happy to contribute the code on Github if there is interest?
>>>>>>> Comments on the handling of feature locations highly welcome!
>>>>>>>
>>>>>>> Frank
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>
>>>>>
>>>>>
>>>>
>>>
>>>
>>>
>>
>


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