How to Obtain Nucleotide Sequence from SeqIO::fastq

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How to Obtain Nucleotide Sequence from SeqIO::fastq

twaddlac
Hello,

     I am curious as to how I am supposed to use SeqIO::fastq to read
in a fastq file and then obtain the nucleotide sequence from that. I
noticed that SeqIO::fastq returns a Seq::Quality object but I haven't
seen a method within that module that returns the nucleotide sequence.
Please, let me know if you have any suggestions!

Thank you very much!!

--

Alan Twaddle, B.S.
MUC class of 2010
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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Roy Chaudhuri-3
Hi Alan,

Another case for the deobfuscator:
http://bioperl.org/cgi-bin/deob_interface.cgi

A Bio::Seq::Quality object is a Bio::PrimarySeq, so you can just say
$seqqual->seq to get the sequence as a string.

Cheers.
Roy.

On 27/07/2010 15:10, Alan Twaddle wrote:

> Hello,
>
>       I am curious as to how I am supposed to use SeqIO::fastq to read
> in a fastq file and then obtain the nucleotide sequence from that. I
> noticed that SeqIO::fastq returns a Seq::Quality object but I haven't
> seen a method within that module that returns the nucleotide sequence.
> Please, let me know if you have any suggestions!
>
> Thank you very much!!
>

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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

twaddlac
On Tue, Jul 27, 2010 at 10:16 AM, Roy Chaudhuri <[hidden email]> wrote:

> Hi Alan,
>
> Another case for the deobfuscator:
> http://bioperl.org/cgi-bin/deob_interface.cgi
>
> A Bio::Seq::Quality object is a Bio::PrimarySeq, so you can just say
> $seqqual->seq to get the sequence as a string.
>
> Cheers.
> Roy.
>
> On 27/07/2010 15:10, Alan Twaddle wrote:
>>
>> Hello,
>>
>>      I am curious as to how I am supposed to use SeqIO::fastq to read
>> in a fastq file and then obtain the nucleotide sequence from that. I
>> noticed that SeqIO::fastq returns a Seq::Quality object but I haven't
>> seen a method within that module that returns the nucleotide sequence.
>> Please, let me know if you have any suggestions!
>>
>> Thank you very much!!
>>
>
>



--

Alan Twaddle, B.S.
MUC class of 2010

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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Roy Chaudhuri-3
In reply to this post by Roy Chaudhuri-3
Hi Alan,

It sounds like there's a problem with reading in your Fastq file. Are
you using the latest BioPerl version? There have been many bug fixes to
Bio::SeqIO::fastq over the last couple of years. If you are using an
up-to-date BioPerl, please could you send an example Fastq entry which
gives the error messages?

Roy.

On 27/07/2010 15:37, Alan Twaddle wrote:

> Whenever I try to access the sequence I get the following error message:
>
>
> --------------------- WARNING ---------------------
> MSG: Seq/Qual descriptions don't match; using sequence description
>
> ---------------------------------------------------
>
> --------------------- WARNING ---------------------
> MSG: Fastq sequence/quality data length mismatch error
>
> ---------------------------------------------------
>
> --------------------- WARNING ---------------------
> MSG: seq doesn't validate with [0-9A-Za-z\*\-\.=~\\/\?], mismatch is +
> ---------------------------------------------------
>
> ------------- EXCEPTION: Bio::Root::Exception -------------
> MSG: Attempting to set the sequence to
> [+GCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTAATGTCGGGAACTCAAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAACTCATCATGCCCCTTGCTGATAC]
> which does not look healthy
> STACK: Error::throw
> STACK: Bio::Root::Root::throw
> /usr/lib/perl5/site_perl/5.8.8/Bio/Root/Root.pm:357
> STACK: Bio::PrimarySeq::seq /usr/lib/perl5/site_perl/5.8.8/Bio/PrimarySeq.pm:270
> STACK: Bio::PrimarySeq::new /usr/lib/perl5/site_perl/5.8.8/Bio/PrimarySeq.pm:221
> STACK: Bio::LocatableSeq::new
> /usr/lib/perl5/site_perl/5.8.8/Bio/LocatableSeq.pm:109
> STACK: Bio::Seq::Meta::Array::new
> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/Meta/Array.pm:167
> STACK: Bio::Seq::Quality::new
> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/Quality.pm:191
> STACK: Bio::Seq::SeqFactory::create
> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/SeqFactory.pm:116
> STACK: Bio::SeqIO::fastq::next_seq
> /usr/lib/perl5/site_perl/5.8.8/Bio/SeqIO/fastq.pm:151
> STACK: fastQParser.pl:12
>
>
>
> On Tue, Jul 27, 2010 at 10:16 AM, Roy Chaudhuri<[hidden email]>  wrote:
>> Hi Alan,
>>
>> Another case for the deobfuscator:
>> http://bioperl.org/cgi-bin/deob_interface.cgi
>>
>> A Bio::Seq::Quality object is a Bio::PrimarySeq, so you can just say
>> $seqqual->seq to get the sequence as a string.
>>
>> Cheers.
>> Roy.
>>
>> On 27/07/2010 15:10, Alan Twaddle wrote:
>>>
>>> Hello,
>>>
>>>       I am curious as to how I am supposed to use SeqIO::fastq to read
>>> in a fastq file and then obtain the nucleotide sequence from that. I
>>> noticed that SeqIO::fastq returns a Seq::Quality object but I haven't
>>> seen a method within that module that returns the nucleotide sequence.
>>> Please, let me know if you have any suggestions!
>>>
>>> Thank you very much!!
>>>
>>
>>
>
>
>

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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

twaddlac
I'm not certain that I'm using the latest BioPerl, but I can check. In
the mean time, I'll send you the example data!

@FQ4HLCS02EO12Q region=2 tag=H
+GCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTAATGTCGGGAACTCAAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAACTCATCATGCCCCTTGCTGATAC
+IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIID666IIIIHHHIIIIIIHHHIIIIIIIIIIDCCHHHIIIIIIGGGIIIIIIIIIIIHHHIIIHH???HHIII???IIIIIIIIIIIIIIIIHHHIIIIIIIIIIIICC@@HIIIIIIIIIIGEGGGGG?4444CCIIIGGGII
@FQ4HLCS02EO12T region=2 tag=B
+GATGAATTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTAAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAACTTCTATCTCTAGAAGGGTCAAAGGATGTCAAGATTTGGTAAGGTTCTTCGCGTTGCAATTCGATGTCGAGC
+DDDGDGGGIIIIIIII@@@@IIIIIIIIIIIIHHB985DFI<<;==DDDGDBDADBG=644466AGB==GDEEFHHEEEHHHHHHHH====GE=D<<<;DBGED;;;;:9955000247<;;;;DGGGGHHHHHBBBHGGGHGGBBB@@@@>;;666EGGEEBBDD<97550//4--,,.62426468=ADD>>6666BBDDEEAEGEG;;;B>@@B;266;;GGDBA?:::995/////9>>9989=9:5


On Tue, Jul 27, 2010 at 10:50 AM, Roy Chaudhuri <[hidden email]> wrote:

> Hi Alan,
>
> It sounds like there's a problem with reading in your Fastq file. Are you
> using the latest BioPerl version? There have been many bug fixes to
> Bio::SeqIO::fastq over the last couple of years. If you are using an
> up-to-date BioPerl, please could you send an example Fastq entry which gives
> the error messages?
>
> Roy.
>
> On 27/07/2010 15:37, Alan Twaddle wrote:
>>
>> Whenever I try to access the sequence I get the following error message:
>>
>>
>> --------------------- WARNING ---------------------
>> MSG: Seq/Qual descriptions don't match; using sequence description
>>
>> ---------------------------------------------------
>>
>> --------------------- WARNING ---------------------
>> MSG: Fastq sequence/quality data length mismatch error
>>
>> ---------------------------------------------------
>>
>> --------------------- WARNING ---------------------
>> MSG: seq doesn't validate with [0-9A-Za-z\*\-\.=~\\/\?], mismatch is +
>> ---------------------------------------------------
>>
>> ------------- EXCEPTION: Bio::Root::Exception -------------
>> MSG: Attempting to set the sequence to
>>
>> [+GCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTAATGTCGGGAACTCAAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAACTCATCATGCCCCTTGCTGATAC]
>> which does not look healthy
>> STACK: Error::throw
>> STACK: Bio::Root::Root::throw
>> /usr/lib/perl5/site_perl/5.8.8/Bio/Root/Root.pm:357
>> STACK: Bio::PrimarySeq::seq
>> /usr/lib/perl5/site_perl/5.8.8/Bio/PrimarySeq.pm:270
>> STACK: Bio::PrimarySeq::new
>> /usr/lib/perl5/site_perl/5.8.8/Bio/PrimarySeq.pm:221
>> STACK: Bio::LocatableSeq::new
>> /usr/lib/perl5/site_perl/5.8.8/Bio/LocatableSeq.pm:109
>> STACK: Bio::Seq::Meta::Array::new
>> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/Meta/Array.pm:167
>> STACK: Bio::Seq::Quality::new
>> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/Quality.pm:191
>> STACK: Bio::Seq::SeqFactory::create
>> /usr/lib/perl5/site_perl/5.8.8/Bio/Seq/SeqFactory.pm:116
>> STACK: Bio::SeqIO::fastq::next_seq
>> /usr/lib/perl5/site_perl/5.8.8/Bio/SeqIO/fastq.pm:151
>> STACK: fastQParser.pl:12
>>
>>
>>
>> On Tue, Jul 27, 2010 at 10:16 AM, Roy Chaudhuri<[hidden email]>
>>  wrote:
>>>
>>> Hi Alan,
>>>
>>> Another case for the deobfuscator:
>>> http://bioperl.org/cgi-bin/deob_interface.cgi
>>>
>>> A Bio::Seq::Quality object is a Bio::PrimarySeq, so you can just say
>>> $seqqual->seq to get the sequence as a string.
>>>
>>> Cheers.
>>> Roy.
>>>
>>> On 27/07/2010 15:10, Alan Twaddle wrote:
>>>>
>>>> Hello,
>>>>
>>>>      I am curious as to how I am supposed to use SeqIO::fastq to read
>>>> in a fastq file and then obtain the nucleotide sequence from that. I
>>>> noticed that SeqIO::fastq returns a Seq::Quality object but I haven't
>>>> seen a method within that module that returns the nucleotide sequence.
>>>> Please, let me know if you have any suggestions!
>>>>
>>>> Thank you very much!!
>>>>
>>>
>>>
>>
>>
>>
>
>



--

Alan Twaddle, B.S.
MUC class of 2010

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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Peter-329
On Tue, Jul 27, 2010 at 3:55 PM, Alan Twaddle <[hidden email]> wrote:

> I'm not certain that I'm using the latest BioPerl, but I can check. In
> the mean time, I'll send you the example data!
>
> @FQ4HLCS02EO12Q region=2 tag=H
> +GCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTAATGTCGGGAACTCAAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAACTCATCATGCCCCTTGCTGATAC
> +IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIID666IIIIHHHIIIIIIHHHIIIIIIIIIIDCCHHHIIIIIIGGGIIIIIIIIIIIHHHIIIHH???HHIII???IIIIIIIIIIIIIIIIHHHIIIIIIIIIIIICC@@HIIIIIIIIIIGEGGGGG?4444CCIIIGGGII
> @FQ4HLCS02EO12T region=2 tag=B
> +GATGAATTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTAAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAACTTCTATCTCTAGAAGGGTCAAAGGATGTCAAGATTTGGTAAGGTTCTTCGCGTTGCAATTCGATGTCGAGC
> +DDDGDGGGIIIIIIII@@@@IIIIIIIIIIIIHHB985DFI<<;==DDDGDBDADBG=644466AGB==GDEEFHHEEEHHHHHHHH====GE=D<<<;DBGED;;;;:9955000247<;;;;DGGGGHHHHHBBBHGGGHGGBBB@@@@>;;666EGGEEBBDD<97550//4--,,.62426468=ADD>>6666BBDDEEAEGEG;;;B>@@B;266;;GGDBA?:::995/////9>>9989=9:5
>

Hopefully this is just an email problem, but that doesn't look like a
well formatted
FASTQ record to me. Where is the "+" line separating the sequence and qualities?

Maybe sending this single record as an attachment would work better (is that
encouraged on this mailing list?).

Peter
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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Fields, Christopher J
On Jul 27, 2010, at 10:34 AM, Peter wrote:

> On Tue, Jul 27, 2010 at 3:55 PM, Alan Twaddle <[hidden email]> wrote:
>> I'm not certain that I'm using the latest BioPerl, but I can check. In
>> the mean time, I'll send you the example data!
>>
>> @FQ4HLCS02EO12Q region=2 tag=H
>> +GCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTAATGTCGGGAACTCAAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAACTCATCATGCCCCTTGCTGATAC
>> +IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIID666IIIIHHHIIIIIIHHHIIIIIIIIIIDCCHHHIIIIIIGGGIIIIIIIIIIIHHHIIIHH???HHIII???IIIIIIIIIIIIIIIIHHHIIIIIIIIIIIICC@@HIIIIIIIIIIGEGGGGG?4444CCIIIGGGII
>> @FQ4HLCS02EO12T region=2 tag=B
>> +GATGAATTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTAAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAACTTCTATCTCTAGAAGGGTCAAAGGATGTCAAGATTTGGTAAGGTTCTTCGCGTTGCAATTCGATGTCGAGC
>> +DDDGDGGGIIIIIIII@@@@IIIIIIIIIIIIHHB985DFI<<;==DDDGDBDADBG=644466AGB==GDEEFHHEEEHHHHHHHH====GE=D<<<;DBGED;;;;:9955000247<;;;;DGGGGHHHHHBBBHGGGHGGBBB@@@@>;;666EGGEEBBDD<97550//4--,,.62426468=ADD>>6666BBDDEEAEGEG;;;B>@@B;266;;GGDBA?:::995/////9>>9989=9:5
>>
>
> Hopefully this is just an email problem, but that doesn't look like a
> well formatted
> FASTQ record to me. Where is the "+" line separating the sequence and qualities?
>
> Maybe sending this single record as an attachment would work better (is that
> encouraged on this mailing list?).
>
> Peter

As long as the attachment isn't many MB in size, I don't have a problem.  Might get delayed by any spam filters though.

chris


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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Dave Messina-3
In reply to this post by Peter-329
> Maybe sending this single record as an attachment would work better (is that
> encouraged on this mailing list?).

Absolutely, please try resending as an attachment, Alan.


Dave


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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

twaddlac
Here's some of the data I would like to parse; the entire file was too
big to send via email. Also, if there is some problem with the data, I
would appreciate any scripting advice since I am unfortunately a noob
:-/

Let me know what you think!

Thank you all very much!

On Tue, Jul 27, 2010 at 12:18 PM, Dave Messina <[hidden email]> wrote:
>> Maybe sending this single record as an attachment would work better (is that
>> encouraged on this mailing list?).
>
> Absolutely, please try resending as an attachment, Alan.
>
>
> Dave
>
>



--

Alan Twaddle, B.S.
MUC class of 2010

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exampleData.fastq (7K) Download Attachment
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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

Roy Chaudhuri-3
Hi Alan,

It looks like there wasn't any problem with the earlier e-mail, and that
your file is not Fastq, but rather some unusual Fastq-like format -
where did you get the file from?

Here's a one-liner to convert your file into standard Fastq:

perl -ne 'print; $_=<>; s/^\+//; print; $_=<>; s/^\+//; print "+\n$_";'
< exampleData.fastq > standard.fastq

I've assumed that your sequences and qualities are all single-line with
a + prefix.

Once you have a standard Fastq file you should be able to parse it with
BioPerl.

Roy.

On 27/07/2010 17:36, Alan Twaddle wrote:

> Here's some of the data I would like to parse; the entire file was too
> big to send via email. Also, if there is some problem with the data, I
> would appreciate any scripting advice since I am unfortunately a noob
> :-/
>
> Let me know what you think!
>
> Thank you all very much!
>
> On Tue, Jul 27, 2010 at 12:18 PM, Dave Messina<[hidden email]>  wrote:
>>> Maybe sending this single record as an attachment would work better (is that
>>> encouraged on this mailing list?).
>>
>> Absolutely, please try resending as an attachment, Alan.
>>
>>
>> Dave
>>
>>
>
>
>
>
>
> _______________________________________________
> Bioperl-l mailing list
> [hidden email]
> http://lists.open-bio.org/mailman/listinfo/bioperl-l

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Re: How to Obtain Nucleotide Sequence from SeqIO::fastq

twaddlac
Ahh thank you very much, everyone!!!!

The data was from a GS-FLX Titanium sequencer but it was parsed out of
a database into the format that was the "incorrect" fastq style.
However, the script worked (though I'm not confident I understand what
it just did) and I have successfully parsed the data out of it via
BioPerl!

Thanks again!!

On Tue, Jul 27, 2010 at 1:14 PM, Roy Chaudhuri <[hidden email]> wrote:

> Hi Alan,
>
> It looks like there wasn't any problem with the earlier e-mail, and that
> your file is not Fastq, but rather some unusual Fastq-like format - where
> did you get the file from?
>
> Here's a one-liner to convert your file into standard Fastq:
>
> perl -ne 'print; $_=<>; s/^\+//; print; $_=<>; s/^\+//; print "+\n$_";' <
> exampleData.fastq > standard.fastq
>
> I've assumed that your sequences and qualities are all single-line with a +
> prefix.
>
> Once you have a standard Fastq file you should be able to parse it with
> BioPerl.
>
> Roy.
>
> On 27/07/2010 17:36, Alan Twaddle wrote:
>>
>> Here's some of the data I would like to parse; the entire file was too
>> big to send via email. Also, if there is some problem with the data, I
>> would appreciate any scripting advice since I am unfortunately a noob
>> :-/
>>
>> Let me know what you think!
>>
>> Thank you all very much!
>>
>> On Tue, Jul 27, 2010 at 12:18 PM, Dave Messina<[hidden email]>
>>  wrote:
>>>>
>>>> Maybe sending this single record as an attachment would work better (is
>>>> that
>>>> encouraged on this mailing list?).
>>>
>>> Absolutely, please try resending as an attachment, Alan.
>>>
>>>
>>> Dave
>>>
>>>
>>
>>
>>
>>
>>
>> _______________________________________________
>> Bioperl-l mailing list
>> [hidden email]
>> http://lists.open-bio.org/mailman/listinfo/bioperl-l
>
>



--

Alan Twaddle, B.S.
MUC class of 2010

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