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<h1>Sequin for Database Submissions and Updates:<br />
A Quick Guide</h1>

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<h2>Introduction</h2>

<p>Sequin is a stand-alone software tool developed by the National
Center for Biotechnology Information (NCBI) for submitting and
updating sequences to the GenBank, EMBL, and DDBJ databases. Sequin
has the capacity to handle long sequences and sets of sequences
(segmented entries, as well as population, phylogenetic, and
mutation studies). It also allows sequence editing and updating,
and provides complex annotation capabilities. In addition, Sequin
contains a number of built-in validation functions for enhanced
quality assurance.</p>

<p>This overview is intended to provide a quick guide to Sequin's
capabilities, including automatic annotation of coding regions, the
graphical viewer, quality control features, and editing features. We
suggest that you read this entire document before beginning your Sequin
submission. More detailed instructions on these and other functions can
be found in Sequin's on-screen <b>Help</b> file, also
available on the World-Wide Web from the Sequin homepage at:</p>

<p><a href=
"http://www.ncbi.nlm.nih.gov/Sequin/">
<tt>http://www.ncbi.nlm.nih.gov/Sequin/</tt></a></p>

<p>Email help is also available from <a href=
"mailto:info@ncbi.nlm.nih.gov">
<tt>info@ncbi.nlm.nih.gov</tt></a></p>


<h2>Table of Contents</h2>

<ul>
<li><a href="#BeforeYouBegin">Before You Begin</a>
<ul>
<li><a href="#PrepareSequenceData">
Preparing Nucleotide and Amino Acid Data</a></li>
<li><a href="#DefinitionLine">Definition Lines</a></li>
<li><a href="#FASTAformat">FASTA Format</a>
<ul>
<li><a href="#SingleSequence">Single Sequence</a></li>
<li><a href="#SegmentedSequences">Segmented Nucleotide Sequences</a></li>
<li><a href="#GappedSequences">Gapped Sequences</a></li>
</ul>
</li>
<li><a href="#AlignmentFormats">Alignment Formats</a>
<ul>
<li><a href="#FASTAplusGAP">FASTA+GAP</a></li>
<li><a href="#PHYLIPformat">PHYLIP</a></li>
<li><a href="#NEXUSInterleaved">NEXUS Interleaved</a></li>
<li><a href="#NEXUSContiguous">NEXUS Contiguous</a></li>
<li><a href="#SetsOfSegmentedSequences">Sets of Segmented Sequences</a></li>
</ul>
</li>
</ul>
</li>
<li><a href="#CreatingASubmission">Creating a Submission</a>
<ul>
<li><a href="#BasicSequinOrganization">Basic Sequin Organization</a></li>
<li><a href="#WelcomeToSequinForm">Welcome to Sequin Form</a></li>
<li><a href="#SubmittingAuthorsForm">Submitting Authors Form</a>
<ul>
<li><a href="#SubmissionPage">Submission Page</a></li>
<li><a href="#ContactPage">Contact Page</a></li>
<li><a href="#AuthorsPage">Authors Page</a></li>
<li><a href="#AffiliationPage">Affiliation Page</a></li>
</ul>
</li>
<li><a href="#SequenceFormatForm">Sequence Format Form</a>
<ul>
<li><a href="#SubmissionType">Submission Type</a></li>
<li><a href="#SequenceDataFormat">Sequence Data Format</a></li>
<li><a href="#SubmissionCategory">Submission Category</a></li>
</ul>
</li>
<li><a href="#OrganismAndSequencesForm">
Organism and Sequences Form</a>
<ul>
<li><a href="#NucleotidePage">Nucleotide Page</a>
<ul>
<li><a href="#NucleotidePageSingleSequence">
Importing Nucleotide FASTA for a Single Sequence</a></li>
<li><a href="#NucleotidePageSequenceSet">
Importing Nucleotide FASTA for a Sequence Set</a></li>
<li><a href="#NucleotidePageAlignment">
Importing an Alignment</a></li>
<li><a href="#AfterImporting">After Importing Files</a></li>
</ul>
</li>
<li><a href="#OrganismPage">Organism Page</a></li>
<li><a href="#ProteinPage">Proteins Page</a></li>
<li><a href="#AnnotationPage">Annotation Page</a></li>
</ul>
</li>
</ul>
</li>
<li><a href="#viewing">Viewing Your Submission</a>
<ul>
<li><a href="#GenBankView">GenBank View</a></li>
<li><a href="#GraphicalView">Graphical View</a></li>
<li><a href="#SequenceView">Sequence View</a></li>
</ul>
</li>
<li><a href="#editing">Editing and Annotating Your Submission</a>
<ul>
<li><a href="#SequenceEditor">Sequence Editor</a></li>
<li><a href="#UpdatingTheSequence">Updating the Sequence</a></li>
<li><a href="#autodefline">Generating the Definition Line</a></li>
<li><a href="#Validation">Record Validation</a></li>
<li><a href="#SubmittingTheEntry">Submitting the Entry</a></li>
</ul>
</li>
<li><a href="#Advanced">Advanced Topics</a>
<ul>
<li><a href="#FeatureEditorDesign">Feature Editor Design</a>
<ul>
<li><a href="#CodingRegionPage">Coding Region Page</a></li>
<li><a href="#PropertiesPage">Properties Page</a></li>
<li><a href="#LocationPage">Location Page</a></li>
</ul>
</li>
<li><a href="#NCBIDesktop">NCBI Desktop</a></li>
<li><a href="#AdditionalInformation">Additional Information</a></li>
</ul>
</li>
<li><a href="#Reference">Reference</a>
<ul>
<li><a href="#NetworkConfiguration">Network Configuration</a></li>
<li><a href="#FeatureTableFormat">Feature Table Format</a></li>
</ul>
</li>
</ul>

<a name="BeforeYouBegin" id="BeforeYouBegin"></a>
<h2>Before You Begin</h2>

<a name="PrepareSequenceData" id = "PrepareSequenceData"></a>
<h3>Preparing Nucleotide and Amino Acid Data</h3>

<p>Sequin normally expects to read sequence files in FASTA format.
Note that most sequence analysis software packages include FASTA or
"raw" as one of the available output formats. Population studies,
phylogenetic studies, mutation studies, and environmental samples
may be entered in either FASTA format, or in PHYLIP, NEXUS, MACAW,
or FASTA+GAP formats if you are submitting an alignment.</p>

<p>See <a href=
"http://www.ncbi.nlm.nih.gov/Sequin/sequin.hlp.html#FASTAFormatforNucleotideSequences">
<tt>http://www.ncbi.nlm.nih.gov/Sequin/sequin.hlp#FASTAFormatforNucleotideSequences</tt></a>
for detailed examples of each of the various input data
formats.</p>

<p>Prepare your sequence data files using a text editor, and save
in ASCII text format (plain text). If your nucleotide sequence
encodes one or more protein products, Sequin expects two files, one
for the nucleotides and one for the proteins.</p>

<a name="DefinitionLine" id="DefinitionLine"></a>
<h3>Definition Lines</h3>

<p>FASTA format is simply the raw sequence preceded by a definition
line. The definition line begins with a &gt; sign and is followed
immediately by a name for the sequence (your own local
identification code, or sequence ID) and a title. During the
submission process, indexing staff at the database to which you are
submitting will change your sequence ID to an Accession number. You
can embed other important information in the title, and Sequin uses
this information to construct a record. Specifically, you can enter
organism and strain or clone information in the nucleotide
definition line and gene and protein information in the protein
definition line using name-value pairs surrounded by square
brackets. Example: [organism=Drosophila melanogaster]
[strain=Oregon R]</p>

<p>Some modifier names have restricted values or formats.</p>

<ul>
<li><b>organism</b> should use the unabbreviated scientific name.
Example: [organism=Drosophila melanogaster]</li>
<li><b>molecule</b> should use either "DNA" or "RNA". Example:
[molecule=DNA]</li>

<li><b>moltype</b> should use one of the following values. Example:
[moltype=genomic]
<ul>
<li>genomic</li>
<li>precursor RNA</li>
<li>mRNA</li>
<li>rRNA</li>
<li>tRNA</li>
<li>snRNA</li>
<li>scRNA</li>
<li>other-genetic</li>
<li>cRNA</li>
<li>snoRNA</li>
<li>transcribed RNA</li>
</ul>
</li>

<li><b>location</b> should use one of the following values.
Example: [location=mitochondrion]
<ul>
<li>genomic</li>
<li>chloroplast</li>
<li>kinetoplast</li>
<li>mitochondrion</li>
<li>plastid</li>
<li>macronuclear</li>
<li>extrachromosomal</li>
<li>plasmid</li>
<li>cyanelle</li>
<li>proviral</li>
<li>virion</li>
<li>nucleomorph</li>
<li>apicoplast</li>
<li>leucoplast</li>
<li>proplastid</li>
<li>endogenous-virus</li>
<li>hydrogenosome</li>
</ul>
</li>

<li><b>collection-date</b> should be in the form YYYY or Mmm-YYYY
or DD-Mmm-YYYY. Example: [collection-date=2005] or
[collection-date=Oct-2005] or
[collection-date=25-Oct-2005]</li>
</ul>

<p>The following modifiers should use only TRUE or FALSE. Example:
[transgenic=TRUE].</p>

<ul>
<li><b>environmental-sample</b></li>
<li><b>germline</b></li>
<li><b>metagenomic</b></li>
<li><b>rearranged</b></li>
<li><b>transgenic</b></li>
</ul>

<p>This is the list of the remaining modifier names that you can
include in your definition lines for nucleotide files:</p>

<table id="sourcemods" summary="remaining modifiers">
<tr>
<td valign="top">
<ul>
<li>acronym</li>
<li>anamorph</li>
<li>authority</li>
<li>bio-material</li>
<li>biotype</li>
<li>biovar</li>
<li>breed</li>
<li>cell-line</li>
<li>cell-type</li>
<li>chemovar</li>
<li>chromosome</li>
<li>clone</li>
<li>clone-lib</li>
<li>collected-by</li>
<li>common</li>
<li>country</li>
<li>cultivar</li>
<li>culture-collection</li>
<li>dev-stage</li>
<li>ecotype</li>
<li>endogenous-virus-name</li>
</ul>
</td>
<td valign="top">
<ul>
<li>forma</li>
<li>forma-specialis</li>
<li>fwd-pcr-primer-name</li>
<li>fwd-pcr-primer-seq</li>
<li>genotype</li>
<li>group</li>
<li>haplotype</li>
<li>identified-by</li>
<li>isolate</li>
<li>isolation-source</li>
<li>lab-host</li>
<li>lat-lon</li>
<li>map</li>
<li>metagenome-source</li>
<li>metagenomic</li>
<li>note</li>
<li>pathovar</li>
<li>plasmid-name</li>
<li>plastid-name</li>
<li>pop-variant</li>
<li>rev-pcr-primer-name</li>
</ul>
</td>
<td valign="top">
<ul>
<li>rev-pcr-primer-seq</li>
<li>segment</li>
<li>serogroup</li>
<li>serotype</li>
<li>serovar</li>
<li>sex</li>
<li>specific-host</li>
<li>specimen-voucher</li>
<li>strain</li>
<li>sub-species</li>
<li>subclone</li>
<li>subgroup</li>
<li>substrain</li>
<li>subtype</li>
<li>synonym</li>
<li>teleomorph</li>
<li>tissue-lib</li>
<li>tissue-type</li>
<li>type</li>
<li>variety</li>
</ul>
</td>
</tr>
</table>

<p>Example: [strain=BALB/c]</p>

<p>Some population studies are a mixture of integrated provirus and
excised virion. These can be indicated by molecule and location
qualifiers, e.g., [molecule=dna] [location=proviral] or
[molecule=rna] [location=virion]. You can also embed
[moltype=genomic] or [moltype=mRNA] to indicate from what source
the molecule was isolated. If you're unsure of which modifier to
use, use [note=...], and database staff will determine the
appropriate modifier to use.</p>

<p>This is the list of modifier names that you can include in your
definition lines for protein files:</p>

<ul>
<li><b>gene</b></li>
<li><b>protein</b></li>
<li><b>prot_desc</b></li>
</ul>

<p>A coding region feature will be created on the nucleotide
sequence indicating where the protein sequence is encoded. If you
specify "gene" in the protein sequence definition line, a gene that
covers the coding region will be created with a locus specified by
the value of "gene".</p>

<p>The product name for the coding region will be the "protein" value
specified in the protein sequence definition line, if supplied. The
product description for the coding region will be the "prot_desc"
value specified in the protein sequence definition line, if
supplied.</p>

<p>Note that the [ and ] brackets actually appear in the text.
(Brackets are sometimes used in computer documentation to denote
optional text. This convention is not followed here.) The bracketed
information will be removed from the definition line for each
sequence. Sequin can also calculate a new definition line by
computing on features in the annotated record (see "<a href=
"#autodefline">Generating the Definition Line</a>").</p>

<p>The ability to embed this information in the definition line is
provided as a convenience to the submitter. If these annotations
are not present, they can be entered in subsequent forms. Sequin is
designed to use this information, and that provided in the initial
forms, to build a properly structured record. <b>In many cases,
the final submission can be completely prepared from these data, so
that no additional manual annotation is necessary once the record
is displayed.</b></p>

<p><b>It is much easier to produce the final submission if you
let Sequin work for you in this manner.</b></p>

<p>In this example we show alternative splicing, where a single
gene produces multiple messenger RNAs that encode two similar but
distinct protein products. Examples for the definition lines for
the nucleotide and protein files are shown here:</p>

<pre>
Nucleotide Sequence:

&gt;eIF4E [organism=Drosophila melanogaster] [strain=Oregon R] Drosophila ...
CGGTTGCTTGGGTTTTATAACATCAGTCAGTGACAGGCATTTCCAGAGTTGCCCTGTTCA ...

Protein Sequences:

&gt;4E-I [gene=eIF4E] [protein=eukaryotic initiation factor 4E-I]
MQSDFHRMKNFANPKSMFKTSAPSTEQGRPEPPTSAAAPAEAKDVKPKEDPQETGEPAGN ...
&gt;4E-II [gene=eIF4E] [protein=eukaryotic initiation factor 4E-II]
MVVLETEKTSAPSTEQGRPEPPTSAAAPAEAKDVKPKEDPQETGEPAGNTATTTAPAGDD ...
</pre>

<p>Also, please note that there must be a line break (carriage
return) between the definition line and the first line of sequence.
Some word processors will break a single line onto two lines
without actually adding a carriage return. (This feature is known
as "word wrapping".) If you are unsure whether there is a carriage
return, you can either set up your word processor so it shows
invisible characters like carriage returns, or view the file in a
text editor that does not create artificial line breaks. <b>The
definition line itself must not have a line break within it,
because the second line would then be misinterpreted as the
beginning of the sequence data.</b> The actual sequence is usually
broken every 50 to 80 characters, but this is not necessary for
Sequin to be able to read it.</p>

<a name="FASTAformat" id="FASTAformat"></a>
<h3>FASTA Format</h3>

<p>There are three types of sequences that may be represented using
the FASTA format: single, contiguous sequences, segmented sequences,
and gapped sequences.</p>

<a name="SingleSequence" id="SingleSequence"></a>
<h4>Single Sequence</h4>

<p>This is the definition line followed by the sequence data. A
sample single sequence file is shown here:</p>

<pre>
&gt;ABC-1 [organism=Saccharomyces cerevisiae][strain=ABC][clone=1]
ATTGCGTTATGGAAATTCGAAACTGCCAAATACTATGTCACCATCATTGA
TGCACCTGGACACAGAGATTTCATCAAGAACATGATCACTGGTACTT
</pre>

<a name="SegmentedSequences" id="SegmentedSequences"></a>
<h4>Segmented Nucleotide Sequences</h4>

<p>A segmented nucleotide entry is an earlier method for capturing
a set of non-contiguous sequences that has a defined order and
orientation. For example, a genomic DNA segmented set could include
encoding exons along with fragments of their flanking introns. An
example of an mRNA segmented pair of records would be the 5' and 3'
ends of an mRNA, where the middle region has not been sequenced. To
import nucleotides in a segmented set, each individual sequence
must be in FASTA format with an appropriate definition line, and
all sequences should be in the same file. Organism information
should only be included in the definition line for the first
segment. Notice that there is a square open bracket on a line by
itself before the first segment and a square close bracket on a
line by itself after the last segment. These square brackets are
required if you are importing multiple segmented sequences, but may
be omitted if you are importing a file that contains all of the
segments and using the "segmented sequence" format. Sequin will
also generate an additional sequence to represent the combination
of the segments, and that sequence will have a distinct sequence
ID. A sample segmented sequence file is shown here:</p>

<pre>
[
&gt;m_gagei_seg1 [organism=Mansonia gagei] Mansonia gagei NADH dehydrogenase ...
ATGGAGCATACATATCAATATTCATGGATCATACCGTTTGTGCCACTTCCAATTCCTATTTTAATAGGAA
TTGGACTCCTACTTTTTCCGACGGCAACAAAAAATCTTCGTCGTATGTGGGCTCTTCCCAATATTTTATT
GTTAAGTATAGTTATGATTTTTTCGGTCGATCTGTCCATTCAGCAAATAAATAAAAGTTCTATCTATCAA
TATGTATGGTCTTGGACCATCAATAATGATTTTTCTTTCGAGTTTGGCTACTTTATTGATTCGCTTACCT
AGTTCGAATTTGATACAAATTTATATTTTTTGGGAATTAGTTGGAATGTGTTCTTATCTATTAATAGGGT
TTTGGTTCACACGACCCGCTGCGGCAAACGCCTGTCAAAAAGCATTTGTAACTAATCGGATAGGCGATTT
TGGTTTATTATTAGGAATCTTAGGTTTTTATTGGATAACGGGAAGTTTCGAATTTCAAGATTTGTTCGAA
ATATTTAATAACTTGATTTATAATAATGAGGTTCAGTTTTTATTTGTTACTTTATGTGCCTCTTTATTA
&gt;m_gagei_seg2
GGTATAATAACAGTATTATTAGGGGCTACTTTAGCTCTTGC
TCAAAAAGATATTAAGAGGGGTTTAGCCTATTCTACAATGTCCCAACTGGGTTATATGATGTTAGCTCTA
GGTATGGGGTCTTATCGAGCCGCTTTATTTCATTTGATTACTCATGCTTATTCGAAGGCATTGTTGTTTT
TAGGATCCGGATCCGTTATTCATTCCATGGAAGCTATTGTTGGATATTCTCCAGATAAAAGCCAGAATAT
GGTTTTTATGGGCGGTTTAAGAAAGCATGTGCCAATTACACAAATTGCTTTTTTAGTGGGTACACTTTCT
CTTTGTGGTATTCCACCCCTTGCTTGTTTTTGGTCCAAAGATGAAATTCTTAGTGACAGCTGGTTGT
&gt;m_gagei_seg3
TCAATAAAACTATGGGGTAAAGAAGAACAAAAAATAATTAACAGAAATTTTCGTTTATCTCCTTTATTAA
TATTAACGATGAATAATAATGAGAAGCCATATAGAATTGGTGATAATGTAAAAAAAGGGGCTCTTATTAC
TATTACGAGTTTTGGCTACAAGAAGGCTTTTTCTTATCCTCATGAATCGGATAATACTATGCTATTTCCT
ATGCTTATATTGGCTCTATTTACTTTTTTTGTTGGAGCCATAGCAATTCCTTTTAATCAAGAAGGACTAC
ATTTGGATATATTATCCAAATTATTAACTCCATCTATAAATCTTTTACATCAAAATTCAAATGATTTTGA
GGATTGGTATCAATTTTTAACAAATGCAACTCTTTCAGTGAGTATAGCCTGTTTCGGAATATTTACAGCA
TTCCTTTTATATAAGCCTTTTTATTCATCTTTACAAAATTTGAACTTACTAAATTTATTTTCGAAAGGGG
GTCCTAAAAGAATTTTTTTGGATAAAATAATATACTTGATATACGATTGGTCATATAATCGTGGTTACAT
AGATACGTTTTATTCAGTATCCTTAACAAAAGGTATAAGAGGATTGGCCGAACTAACTCATTTTTTTGAT
AGGCGAGTAATCGATGGAATTACAAATGGAGTACGCATCACAAGTTTTTTTATAGGCGAAGGTATCAAAT
ATT
]
</pre>

<a name="GappedSequences" id="GappedSequences"></a>
<h4>Gapped Sequences</h4>

<p>A gapped sequence represents a newer method for describing
non-contiguous sequences, but only requires a single sequence
identifier. A gap is represented by a line that starts with &gt;?
and is immediately followed by either a length (for gaps of known
length) or "unk100" for gaps of unknown length. For example,
"&gt;?200". The next sequence segment continues on the next line,
with no separate definition line or identifier. The difference
between a gapped sequence and a segmented sequence is that the
gapped sequence uses a single identifier and can specify known
length gaps. Gapped sequences are preferred over segmented
sequences. A sample gapped sequence file is shown here:</p>

<pre>
&gt;m_gagei [organism=Mansonia gagei] Mansonia gagei NADH dehydrogenase ...
ATGGAGCATACATATCAATATTCATGGATCATACCGTTTGTGCCACTTCCAATTCCTATTTTAATAGGAA
TTGGACTCCTACTTTTTCCGACGGCAACAAAAAATCTTCGTCGTATGTGGGCTCTTCCCAATATTTTATT
GTTAAGTATAGTTATGATTTTTTCGGTCGATCTGTCCATTCAGCAAATAAATAAAAGTTCTATCTATCAA
TATGTATGGTCTTGGACCATCAATAATGATTTTTCTTTCGAGTTTGGCTACTTTATTGATTCGCTTACCT
AGTTCGAATTTGATACAAATTTATATTTTTTGGGAATTAGTTGGAATGTGTTCTTATCTATTAATAGGGT
TTTGGTTCACACGACCCGCTGCGGCAAACGCCTGTCAAAAAGCATTTGTAACTAATCGGATAGGCGATTT
TGGTTTATTATTAGGAATCTTAGGTTTTTATTGGATAACGGGAAGTTTCGAATTTCAAGATTTGTTCGAA
ATATTTAATAACTTGATTTATAATAATGAGGTTCAGTTTTTATTTGTTACTTTATGTGCCTCTTTATTA
&gt;?200
GGTATAATAACAGTATTATTAGGGGCTACTTTAGCTCTTGC
TCAAAAAGATATTAAGAGGGGTTTAGCCTATTCTACAATGTCCCAACTGGGTTATATGATGTTAGCTCTA
GGTATGGGGTCTTATCGAGCCGCTTTATTTCATTTGATTACTCATGCTTATTCGAAGGCATTGTTGTTTT
TAGGATCCGGATCCGTTATTCATTCCATGGAAGCTATTGTTGGATATTCTCCAGATAAAAGCCAGAATAT
GGTTTTTATGGGCGGTTTAAGAAAGCATGTGCCAATTACACAAATTGCTTTTTTAGTGGGTACACTTTCT
CTTTGTGGTATTCCACCCCTTGCTTGTTTTTGGTCCAAAGATGAAATTCTTAGTGACAGCTGGTTGT
&gt;?unk100
TCAATAAAACTATGGGGTAAAGAAGAACAAAAAATAATTAACAGAAATTTTCGTTTATCTCCTTTATTAA
TATTAACGATGAATAATAATGAGAAGCCATATAGAATTGGTGATAATGTAAAAAAAGGGGCTCTTATTAC
TATTACGAGTTTTGGCTACAAGAAGGCTTTTTCTTATCCTCATGAATCGGATAATACTATGCTATTTCCT
ATGCTTATATTGGCTCTATTTACTTTTTTTGTTGGAGCCATAGCAATTCCTTTTAATCAAGAAGGACTAC
ATTTGGATATATTATCCAAATTATTAACTCCATCTATAAATCTTTTACATCAAAATTCAAATGATTTTGA
GGATTGGTATCAATTTTTAACAAATGCAACTCTTTCAGTGAGTATAGCCTGTTTCGGAATATTTACAGCA
TTCCTTTTATATAAGCCTTTTTATTCATCTTTACAAAATTTGAACTTACTAAATTTATTTTCGAAAGGGG
GTCCTAAAAGAATTTTTTTGGATAAAATAATATACTTGATATACGATTGGTCATATAATCGTGGTTACAT
AGATACGTTTTATTCAGTATCCTTAACAAAAGGTATAAGAGGATTGGCCGAACTAACTCATTTTTTTGAT
AGGCGAGTAATCGATGGAATTACAAATGGAGTACGCATCACAAGTTTTTTTATAGGCGAAGGTATCAAAT
ATT
</pre>

<a name="AlignmentFormats" id="AlignmentFormats"></a>
<h3>Alignment Formats</h3>

<p>Once you have created your alignment file, be sure to note the
characters used to indicate ambiguous bases, bases that match the
master sequence, and gaps in the alignment. Be aware that some
alignment formats use different characters to indicate gaps used to
pad sequences at the beginning, middle, and end of the alignment.
You will be able to specify these characters separately before
importing the alignment file.</p>

<a name="FASTAplusGAP" id="FASTAplusGAP"></a>
<h4>FASTA+GAP</h4>

<pre>
&gt;ABC-1 [organism=Saccharomyces cerevisiae][strain=ABC][clone=1]
---ATTGCGTTATGGAAATTCGAAACTGCCAAATACTATGTCACCATCAT
TGATGCACCTGGACACAGAGATTTCATCAAGAACATGATCACTGGTACTT
&gt;ABC-2 [organism=Saccharomyces cerevisiae][strain=ABC][clone=2]
GATATTGCTTTATGGAAATTCGAAACTGCCAAATACTATGTCACCATCAT
TGATGCACCTGGACACAGAAATTTCATCAAGAACATGATCACTGGTACTT
&gt;ABC-3 [organism=Saccharomyces cerevisiae][strain=ABC][clone=3]
---ATTGCTTTATGGAAATTCGAAACTGCCAAATACTATGTTA-------
TGATGCACCTGGACACAGAGATTTCATCAAAAACATGATCACTGGTACTT
</pre>

<a name="PHYLIPformat" id="PHYLIPformat"></a>
<h4>PHYLIP</h4>

<pre>
      3  100
ABC-1      ---ATTGCGT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
ABC-2      GATATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
ABC-3      ---ATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TTA-------

           TGATGCACCT GGACACAGAG ATTTCATCAA GAACATGATC ACTGGTACTT
           TGATGCACCT GGACACAGAA ATTTCATCAA GAACATGATC ACTGGTACTT
           TGATGCACCT GGACACAGAG ATTTCATCAA AAACATGATC ACTGGTACTT

&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=1]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=2]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=3]
</pre>

<a name="NEXUSInterleaved" id="NEXUSInterleaved"></a>
<h4>NEXUS Interleaved</h4>

<pre>
#NEXUS

begin data;
        dimensions  ntax=3 nchar=100;
        format datatype=dna  missing=? gap=-  interleave ;
        matrix

[     1                                                   50]
ABC_1 ???ATTGCGT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
ABC_2 GATATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
ABC_3 ???ATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TTA-------

[     51                                                 100]
ABC_1 TGATGCACCT GGACACAGAG ATTTCATCAA GAACATGATC ACTGGTACTT
ABC_2 TGATGCACCT GGACACAGAA ATTTCATCAA GAACATGATC ACTGGTACTT
ABC_3 TGATGCACCT GGACACAGAG ATTTCATCAA AAACATGATC ACTGGTACTT
;
END;

begin ncbi;
sequin
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=1]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=2]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=3]
;
end;
</pre>

<a name="NEXUSContiguous" id="NEXUSContiguous"></a>
<h4>NEXUS Contiguous</h4>

<pre>
#NEXUS

begin data;
        dimensions  ntax=3 nchar=100;
        format datatype=dna  missing=? gap=-  ;
        matrix

ABC_1   
???ATTGCGT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
TGATGCACCT GGACACAGAG ATTTCATCAA GAACATGATC ACTGGTACTT
ABC_2  
GATATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TCACCATCAT
TGATGCACCT GGACACAGAA ATTTCATCAA GAACATGATC ACTGGTACTT
ABC_3  
???ATTGCTT TATGGAAATT CGAAACTGCC AAATACTATG TTA-------
TGATGCACCT GGACACAGAG ATTTCATCAA AAACATGATC ACTGGTACTT
;
END;

begin ncbi;
sequin
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=1]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=2]
&gt;[organism=Saccharomyces cerevisiae][strain=ABC][clone=3]
;
end;
</pre>

<a name="SetsOfSegmentedSequences" id="SetsOfSegmentedSequences"></a>
<h4>Sets of Segmented Sequences</h4>

<p>If the sequences in a phylogenetic study are really segmented
(e.g., exons 2 and 3 of a gene without intron 2), the individual
segments from a single organism can be grouped within square
brackets. Subsequent segments are detected by the presence of a
FASTA definition line. For example:</p>

<pre>
[
&gt;Qruex2 [organism=Quercus rubra]
CGAAAACCTGCACAGCAGAAACGACTCGCAAACTAGTAATAACTGACGGAGGACGGAGGG ...
&gt;Qruex3
CATCATTGCCCCCCATCCTTTGGTTTGGTTGGGTTGGAAGTTCACCTCCCATATGTGCCC ...
]
[
&gt;Qsuex2 [organism=Quercus suber]
CAAACCTACACAGCAGAACGACTCGAGAACTGGTGACAGTTGAGGAGGGCAAGCACCTTG ...
&gt;Qsuex3
CATCGTTGCCCCCCTTCTTTGGTTTGGTTGGGTTGGAAGTTGGCCTTCCATATGTGCCCT ...
]
...
</pre>

<p>FASTA+GAP format can also use this convention for encoding sets
of aligned segmented sequences.</p>

<a name="CreatingASubmission" id="CreatingASubmission"></a>
<h2>Creating a Submission</h2>

<p>The sequence data we will use for this example is the genomic
sequence of the <span class="taxonomy">Drosophila melanogaster</span>
eukaryotic initiation factors 4E-I and 4E-II (GenBank Accession number
U54469).</p>

<a name="BasicSequinOrganization" id="BasicSequinOrganization"></a>
<h3>Basic Sequin Organization</h3>

<p>Sequin is organized into a series of forms for entering
submitting authors, entering organism and sequences, entering
information such as strain, gene, and protein names, viewing the
complete submission, and editing and annotating the submission. The
goal is to go quickly from raw sequence data to an assembled record
that can be viewed, edited, and submitted to your database of
choice.</p>

<p>Advance through the pages that make up each form by clicking on
labeled folder tabs or the <span class="buttonlabel">Next Page</span>
button. After the basic information forms have been completed and the
sequence data imported, Sequin provides a complete view of your
submission, in your choice of text or graphic format. At this point,
any of the information fields can be easily modified by double-clicking
on any area of the record, and additional biological annotations can be
entered by selecting from a menu.</p>

<p>Sequin has an on-screen <span class="buttonlabel">Help</span> file
that is opened automatically when you start the program. Because it is
context sensitive, the <span class="buttonlabel">Help</span> text will
change and follow your steps as you progress through the program. A
"Find" function is also provided.</p>

<a name="WelcomeToSequinForm" id="WelcomeToSequinForm"></a>
<h3>Welcome to Sequin Form</h3>

<p><img class="figure" src="images/welcome.png" alt=
"Welcome to Sequin Form" /></p>

<p>Once you have finished preparing the sequence files, you are
ready to start the Sequin program. Sequin's first window asks you
to indicate the database to which the sequence will be submitted
and prompts you to start a new project or continue with an existing
one. Once you choose a database, Sequin will remember it in
subsequent sessions. In general, each sequence submission should be
entered as a separate project. However, segmented DNA sequences,
gapped sequences, population studies, phylogenetic studies, and
mutation studies should be submitted together as one project. This
feature also eliminates the need to save Sequin information
templates for each sequence.</p>

<p>To begin creating your submission, click the <span
class="buttonlabel">Start New Submission</span> button.</p>

<a name="SubmittingAuthorsForm" id="SubmittingAuthorsForm"></a>
<h3>Submitting Authors Form</h3>

<p>The pages in the <span class="dialoglabel">Submitting Authors</span>
form ask you to provide the release date, a working title, names and
contact information of submitting authors, and affiliation information.
To create a personal template for use in future submissions, use the
<span class="menulabel">File-&gt;Export</span> menu item after
completing each page of this form.</p>

<a name="SubmissionPage" id="SubmissionPage"></a>
<h4>Submission Page</h4>

<p><img class="figure" src="images/submit.png" alt=
"Submission Page" /></p>

<p>The <span class="folderlabel">Submission</span> page asks for a
tentative title for a manuscript describing the sequence and will
initially mark the manuscript as being unpublished. When the article is
published, the database staff will update the sequence record with the
new citation. This page also lets you indicate that a record should be
held confidential by the database until a specified date, although the
preferred policy is to release the record immediately into the public
databases.</p>

<a name="ContactPage" id="ContactPage"></a>
<h4>Contact Page</h4>

<p><img class="figure" src="images/contact.png" alt=
"Contact Page" /></p>

<p>The <span class="folderlabel">Contact</span> page asks for the name,
phone number, and email address of the person responsible for making
the submission. Database staff members will contact this person if
there are any questions about the record.</p>

<p>The Sfx (suffix) popup is used to enter personal name suffixes
(e.g., Jr., Sr., or III), not a person's academic degrees (e.g., MD
or PhD). Also, it is not necessary to type periods after
initials.</p>

<a name="AuthorsPage" id="AuthorsPage"></a>
<h4>Authors Page</h4>

<p><img class="figure" src="images/authors.png" alt=
"Authors Page" /></p>

<p>In the <span class="folderlabel">Authors</span> page, enter the
names of the people who should get scientific credit for the sequence
presented in this record. These will become the authors for the initial
(unpublished) manuscript.</p>

<p>Authors are entered in a spreadsheet. As soon as anything is
typed in the last row, a new (blank) row is added below it. Use the
tab key to move between fields. Tabbing from the last column
automatically moves to the First Name column in the next row.</p>

<a name="AffiliationPage" id="AffiliationPage"></a>
<h4>Affiliation Page</h4>

<p><img class="figure" src="images/affil.png" alt=
"Affiliation Page" /></p>

<p>The <span class="folderlabel">Affiliation</span> page asks for the
institutional affiliation of the primary author.</p>

<a name="SequenceFormatForm" id="SequenceFormatForm"></a>
<h3>Sequence Format Form</h3>

<p><img class="figure" src="images/format.png" alt=
"Format Form" /></p>

<p>With Sequin, the actual sequence data are imported from an
outside data file. So before you begin, prepare your sequence data
files using a text editor, perhaps one associated with your
laboratory sequence analysis software (see "<a href=
"#BeforeYouBegin">Before you Begin</a>").</p>

<a name="SubmissionType" id="SubmissionType"></a>
<h4>Submission Type</h4>

<p>If you have sequence data from a single source, choose from one of
the following submission types:</p>

<ul>
<li><span class="buttonlabel">Single Sequence</span> if you have a
single contiguous mRNA or genomic DNA sequence.</li>
<li><span class="buttonlabel">Segmented Sequence</span> if you have a
single collection of non-overlapping, non-contiguous sequences that
cover a specified genetic region from a single source. A standard
example is a set of genomic DNA sequences that encode exons from a gene
along with fragments of their flanking introns.</li>
<li><span class="buttonlabel">Gapped Sequence</span> if you have a
single non-contiguous mRNA or genomic DNA sequence. A gapped sequence
contains specified gaps of known or unknown length where the exact
nucleotide sequence has not been determined.</li>
</ul>

<p>See <a href="#BeforeYouBegin">Before You Begin</a> if you have
questions about how to format your files or about the differences
between these formats.</p>

<p>If you have a set of single sequences, segmented sequences, or
gapped sequences or a mixture of these types of sequences, you will
need to choose one of the following submission types:</p>

<ul>
<li><span class="buttonlabel">Population Study</span> for a set derived
by sequencing the same gene from different isolates of the same
organism.</li>
<li><span class="buttonlabel">Phylogenetic Study</span> for a set
derived by sequencing the same gene from different organisms.</li>
<li><span class="buttonlabel">Mutation Study</span> for a set derived
by sequencing multiple mutations of a single gene.</li>
<li><span class="buttonlabel">Environmental Samples</span> for a set
derived by sequencing the same gene from a population of unclassified
or unknown organisms.</li>
<li><span class="buttonlabel">Batch Submission</span> for a set that is
not a population study, mutation study, phylogenetic study, or
environmental samples. The sequences should be related in some way,
such as coming from the same publication or organism. You should plan
that all sequences will be released to the public on the same date.</li>
</ul>

<a name="SequenceDataFormat" id="SequenceDataFormat"></a>
<h4>Sequence Data Format</h4>

<p>If you have chosen <span class="buttonlabel">Single Sequence</span>,
<span class="buttonlabel">Segmented Sequence</span>, <span
class="buttonlabel">Gapped Sequence</span>, or <span
class="buttonlabel">Batch Submission</span> for the submission type,
you will only be able to select <span class="buttonlabel">FASTA (no
alignment).</span></p>

<p>If you have chosen one of the other submission types, you may import
the sequences in FASTA format, or you may choose to import the
sequences using an alignment file by selecting
<span class="buttonlabel">Alignment (FASTA+GAP, NEXUS, PHYLIP, etc.)</span>.
See <a href= "#AlignmentFormats">Alignment Formats</a> for an
explanation of the available formats for alignment files.</p>

<a name="SubmissionCategory" id="SubmissionCategory"></a>
<h4>Submission Category</h4>

<p>Choose <span class="buttonlabel">Original Submission</span> if you
have directly sequenced the nucleotide sequence in your laboratory.</p>

<p>Choose <span class="buttonlabel">Third Party Annotation</span> if
you have downloaded or assembled sequence from GenBank and modified it
with your own annotations. See <a href=
"http://www.ncbi.nih.gov/Genbank/TPA.html">
<tt>http://www.ncbi.nih.gov/Genbank/TPA.html</tt></a> for more information
about Third Party Annotation rules.</p>

<a name="OrganismAndSequencesForm" id="OrganismAndSequencesForm"></a>
<h3>Organism and Sequences Form</h3>

<p>The <span class="dialoglabel">Organism and Sequences</span> form has
been enhanced with a number of Assistants that allow entry or editing
of sequence and source information.</p>

<a name="NucleotidePage" id="NucleotidePage"></a>
<h4>Nucleotide Page</h4>

<p>The <span class="folderlabel">Nucleotide</span> page will have one of
three appearances, based on whether you have chosen to import a single
sequence, a set of sequences, or an alignment.</p>

<a name="NucleotidePageSingleSequence" id="NucleotidePageSingleSequence"></a>
<h5>Importing Nucleotide FASTA for a Single Sequence</h5>

<p><img class="figure" src="images/nucsing1.png" alt=
"Single Sequence Page" /></p>

<p>To import a single sequence, click on <span
class="buttonlabel">Import Nucleotide FASTA</span> and enter the name
of the file that contains your FASTA sequence. See
<a href="http://www.ncbi.nlm.nih.gov/Sequin/QuickGuide/sequin.htm#BeforeYouBegin">
Before You Begin</a> for information on how to format your FASTA file.
In addition to importing from a file, sequences can also be read by
pasting from the computer's "clipboard" using the <span
class="menulabel">Edit-&gt;Paste</span> menu item or by using the <span
class="buttonlabel">Add/Modify Sequences</span> button.</p>

<a name="NucleotidePageSequenceSet" id="NucleotidePageSequenceSet"></a>
<h5>Importing Nucleotide FASTA for a Sequence Set</h5>

<p><img class="figure" src="images/nucset.png" alt=
"Sequence Set Page" /></p>

<p>To import a set of sequences, click on <span
class="buttonlabel">Import Nucleotide FASTA</span> and enter the name
of the file that contains some or all of your FASTA sequences. See
<a href="http://www.ncbi.nlm.nih.gov/Sequin/QuickGuide/sequin.htm#BeforeYouBegin">
Before You Begin</a> for information on how to format your FASTA file.
You may click on <span class="buttonlabel">Import Additional Nucleotide
FASTA</span> to import additional files if your sequences are in more
than one file. In addition to importing from a file, sequences can also
be read by pasting from the computer's "clipboard" using the <span
class="menulabel">Edit-&gt;Paste menu</span> item or by using the <span
class="buttonlabel">Add/Modify Sequences</span> button.</p>

<p>If you would like to create an alignment for your set of sequences,
check <span class="buttonlabel">Create Alignment</span> on this page.</p>

<a name="NucleotidePageAlignment" id="NucleotidePageAlignment"></a>
<h5>Importing an Alignment</h5>

<p><img class="figure" src="images/nucaln.png" alt=
"Importing an Alignment" /></p>

<p>See <a href=
"http://www.ncbi.nlm.nih.gov/Sequin/QuickGuide/sequin.htm#BeforeYouBegin">
Before You Begin</a> for information on how to format your
alignment file. Before importing your alignment, choose which
characters in the alignment file represent gaps, ambiguous or
unknown nucleotides, and "matches".</p>

<p>Some data files distinguish between gaps at the beginning, in the
middle, and at the end of a sequence. These characters can be
entered separately if needed, or you may specify the same character
for all three kinds of gaps if appropriate.</p>

<p><span class="textlabel">Ambiguous/Unknown</span> characters
represent nucleotides that are present in the sequence but were not
sequenced. Usually this is "N". <span class="textabel">Match</span>
characters are characters in a sequence other than the first that match
the character at that alignment position in the first sequence. When
match characters are used, usually they are specified as ".", but when
match characters are not used, "." is frequently used as a gap
character, so the ":" is supplied instead as a default.</p>

<p>You may specify more than one character for each of these
categories. When you have filled out the character information, click
on <span class="buttonlabel">Import Nucleotide Alignment</span> and
enter the name of your alignment file.</p>

<a name="AfterImporting" id="AfterImporting"></a>
<h5>After Importing Files</h5>

<p><img class="figure" src="images/nucsing2.png" alt=
"After Importing Files" /></p>

<p>When the sequence file or alignment file import is complete, a box
will appear showing the number of nucleotide segments imported, the
total length in nucleotides of the sequences entered, and the sequence
ID(s) you designated. The actual sequence data are <b>not</b>
shown. If any of this information is missing or incorrect, check the
file containing the sequence data for proper FASTA format, click on the
<span class="buttonlabel">Clear Sequences</span> button, then reimport
the sequence(s).</p>

<p>If the imported nucleotide sequence or sequences or alignment
have any problems, such as colliding local identifiers in a set or
mismatched brackets in the definition line, an Assistant dialog
appears to help correct the problems. Severe problems must be fixed
before you can continue with the Sequin submission.</p>

<a name="OrganismPage" id="OrganismPage"></a>
<h4>Organism Page</h4>

<p><img class="figure" src="images/organism.png" alt=
"Organism Page" /></p>

<p>The second page of the <span class="folderlabel">Organism and
Sequences</span> form requests information regarding the scientific
name of the organism from which the sequence was derived, if it was not
already encoded in the nucleotide FASTA file. There are Assistants for
manually adding organism name information or adding source
qualifiers.</p>

<p>Sequin has extracted the organism and strain names from the FASTA
definition line in this example, eliminating the need to manually enter
information in the <span class="folderlabel">Organism</span> page.</p>

<a name="ProteinPage" id="ProteinPage"></a>
<h4>Proteins Page</h4>

<p><img class="figure" src="images/protein1.png" alt=
"Proteins Page" /></p>

<p>If your sequence or sequences encode one or more proteins, you can
enter the sequences of the protein products in this page. To import the
amino acid sequences, click on the <span
class="folderlabel">Proteins</span> folder tab and click on the <span
class="buttonlabel">Import Protein FASTA</span> button. You may import
more than one file by clicking the button again after importing the
first file. See <a href="#BeforeYouBegin">Before You Begin</a> for
information on how to format your protein files.</p>

<p><img class="figure" src="images/protein2.png" alt=
"Proteins Example" /></p>

<p>In this example, we imported two protein sequences. These are
the alternative splice products of the same gene. Both protein
sequences were in the same data file, but each had its own
definition line.</p>

<p>Sequin has extracted the gene and protein names from the FASTA
definition lines, and will use these to construct the initial
sequence record.</p>

<a name="AnnotationPage" id="AnnotationPage"></a>
<h4>Annotation Page</h4>

<p><img class="figure" src="images/annot.png" alt=
"Annotation Page" /></p>

<p>The <span class="folderlabel">Annotation</span> page allows you to
add an rRNA or CDS feature to the entire length of all sequences in the
set. In addition, you can add a title to any sequences that didn't
obtain them from a FASTA definition line. It is much easier to add
these in bulk at this step than to add individual rRNA or CDS features
to each sequence after the record is constructed.</p>

<p>It is customary in a nucleotide record to format titles for
sequences containing coding region features in the following
way:</p>

<p>Genus species protein name (gene symbol) mRNA/gene,
complete/partial cds.</p>

<p>The choice of "mRNA" or "gene" depends upon the molecule type (use
"mRNA" for mRNA or cDNA, and "gene" for genomic DNA). Use "partial" for
incomplete features. The proper organism name in a phylogenetic study
can be added to the beginning of each title automatically by checking
the <span class="buttonlabel">Prefix title with organism name</span>
box.</p>

<p>However, for records containing CDS, rRNA, or tRNA features,
Sequin can generate the definition line automatically by computing
on the features (see "<a href="#autodefline">Generating the
Definition Line</a>").</p>

<p>More complex situations, such as a population study of HIV
sequences, can include multiple CDS features in each sequence. In this
case, do not use the <span class="folderlabel">Annotation</span> page
to create features. (You can still use it for a common title, however.)
After the initial submission has been created, you would manually
annotate features onto one of the sequences. If you are submitting an
alignment, or if you are submitting a set of sequences and you have
checked <span class="buttonlabel">Create Alignment</span> on the
<span class="folderlabel">Nucleotide</span> page, you will be able to
use feature propagation to annotate the same features at the equivalent
aligned locations on the remaining sequences.</p>

<a name="viewing" id="viewing"></a>
<h2>Viewing Your Submission</h2>

<a name="GenBankView" id="GenBankView"></a>
<h3>GenBank View</h3>

<p>After you have completed importing the data files, Sequin will
display your full submission information in the GenBank format (or
EMBL format if you chose EMBL as the database for submission in the
first form).</p>

<p><img class="figure" src="images/genbank.png" alt=
"GenBank Format" /></p>

<p>On the basis of the information provided in your DNA and amino
acid sequence files, any coding regions will be automatically
identified and annotated for you. The figure shows only the top
portion of the GenBank record, but you can see the first of two
coding region (CDS) features. The vertical bar to the left of the
paragraph indicates that the CDS has been selected by clicking with
the computer's mouse.</p>

<p>You may now make changes to the coding region, publication, source,
and other features in the record by double clicking on the appropriate
paragraphs in the GenBank display format. You may also use the <span
class="menulabel">Annotate-&gt;Generate Definition Line</span> menu
item to <a href="#autodefline">compute a definition line</a> for the
annotated features in the record.</p>

<a name="GraphicalView" id="GraphicalView"></a>
<h3>Graphical View</h3>

<p><img class="figure" src="images/graphic.png" alt=
"Graphic Format" /></p>

<p>To get a graphical view, change the <span
class="popuplabel">Format</span> popup menu from <span
class="menulabel">GenBank</span> to <span
class="menulabel">Graphic</span>. Reviewing your submission in Graphic
format allows you to visually confirm expected location of exons,
introns, and other features in multiple interval coding regions. The
Graphic view in our eukaryotic initiation factor example illustrates
how the coding region intervals for the two protein products are
spatially related to each other.</p>

<p>The <span class="menulabel">File-&gt;Duplicate View</span> menu item
will launch a second viewer on the record. The display format on each
viewer can be independently set, allowing you to see a graphical view
and a GenBank text report simultaneously. This is useful for getting an
overall view of the features and seeing the details of annotation.</p>

<a name="SequenceView" id="SequenceView"></a>
<h3>Sequence View</h3>

<p><img class="figure" src="images/sequence.png" alt=
"Sequence Format" /></p>

<p>Sequence view is a static version of the sequence and alignment
editor. It shows the actual nucleotide sequence, with feature
intervals annotated directly on the sequence. Protein translations
of CDS features are also shown, as are all features shown in the
graphical view.</p>

<a name="editing" id="editing"></a>
<h2>Editing and Annotating Your Submission</h2>

<p>At this point, Sequin could process your entry based on what you
have entered so far, and you could send it to your nucleotide database
of choice (as set in the initial form). However, to optimize the
usefulness of your entry for the scientific community, you may want to
provide additional information to indicate biologically significant
regions of the sequence. But first, save the entry so that if you make
any unwanted changes during the editing process you can revert to the
original copy.</p>

<p>Additional information may be in the form of Descriptors or
Features. Descriptors are annotations that apply to an entire
sequence or set of sequences. They are used to remove redundant
information in a record. Features are annotations that apply to a
specific sequence interval.</p>

<p>Sequin provides two methods to modify your entry: (1) to edit
existing information, double click on the text or graphic area you want
to modify, and Sequin will display forms requesting needed information;
or (2) to add new information, use the <span
class="menulabel">Annotate</span> menu and select from the list of
available annotations.</p>

<a name="SequenceEditor" id="SequenceEditor"></a>
<h3>Sequence Editor</h3>

<p>Additional sequence data can also be added using Sequin's sequence
editor, which can be launched using the <span
class="menulabel">Edit-&gt;Edit Sequence</span> menu item. Sequin will
automatically adjust feature intervals when editing the sequence. Prior
to Sequin, it was usually easier to reannotate everything from scratch
when the sequence changed. But an even easier way to update sequences
is described in the following section.</p>

<a name="UpdatingTheSequence" id="UpdatingTheSequence"></a>
<h3>Updating the Sequence</h3>

<p>Sequin can also read in a replacement sequence, or an
overlapping sequence extension, and perform the alignment and
feature propagation calculations necessary to adjust feature
intervals, even though the individual editing operations were not
done with the sequence editor.</p>

<p>The <span class="menulabel">Edit-&gt;Update Sequence</span> submenu
has several choices. These are for use by the original submitter of a
record.</p>

<p>You can read a FASTA file or raw sequence file. This can be a
replacement sequence, or it can overlap the original sequence at
the 5' or 3' end. After Sequin aligns the two sequences, and you
select optional parameters, the sequence in your record is updated,
with all feature intervals adjusted properly.</p>

<p>You can also update with an existing sequence record that
contains features. This can be obtained from a file, or retrieved
from Entrez either via an Accession number. The latter choice
requires the <a href=
"http://www.ncbi.nlm.nih.gov/Sequin/netaware.html">network-aware</a>
version of Sequin. Once it gets the new record, Sequin aligns the
two sequences as before. This is typically used either to merge two
records that overlap, or to copy features from database records
onto a new large contig.</p>

<p><img class="figure" src="images/update.png" alt=
"Update Sequence Form" /></p>

<p>The first panel shows how the two sequences align to each other.
In this case, it is a 5' extension of the existing sequence. 400
bases are new, 70 bases overlap the old sequence, and there are 30
bases of vector on the new sequence that do not align to the old
sequence and will be trimmed off.</p>

<p>The second panel shows details of the 70-base aligned region.
There is one single base gap in each sequence. The total number of
sequence letters plus gap characters is the alignment length, 71 in
this example. (This number was shown between the sequence figures
in the first panel.) Mismatched bases are indicated by vertical red
lines between the two sequences.</p>

<p>The third panel shows the actual sequence letters in the aligned
region. Clicking on a gap or mismatch in the second panel scrolls
to the appropriate place in this panel.</p>

<p>Before pressing <span class="buttonlabel">Update Sequence</span>,
you need to enter optional parameters. The alignment relationship is
calculated by Sequin, but in some cases you may want to replace or
patch rather than extend the existing sequence.</p>

<a name="autodefline" id="autodefline"></a>
<h3>Generating the Definition Line</h3>

<p>The <span class="menulabel">Annotate-&gt;Generate Definition
Line</span> menu item can make the appropriate titles once the record
has been annotated with features. The general format for sequences
containing coding region features is:</p>

<p>Genus species protein name (gene symbol) mRNA/gene,
complete/partial cds.</p>

<p>Exceptional cases, where this automatic function is unable to
generate a reasonable definition line, will be edited by the
database staff to conform to the style conventions.</p>

<p>The new definition line will replace any previous title,
including that originally on the FASTA definition line.</p>

<a name="Validation" id="Validation"></a>
<h3>Record Validation</h3>

<p>Once you are satisfied that you have entered all the relevant
information, save your file! Then select the <span
class="menulabel">Search-&gt;Validate</span> menu item. You will either
receive a message that the validation test succeeded or see a screen
listing the validation errors and warnings. Just double click on an
error item to launch the appropriate editor for making corrections. The
validator includes checks for such things as missing organism
information, incorrect coding region lengths, internal stop codons in
coding regions, inconsistent genetic codes, mismatched amino acids, and
non-consensus splice sites.</p>

<p><img class="figure" src="images/validate.png" alt=
"Record Validator Form" /></p>

<a name="SubmittingTheEntry" id="SubmittingTheEntry"></a>
<h3>Submitting the Entry</h3>

<p>When the entry is properly formatted and error-free, click the <span
class="buttonlabel">Done</span> button or select the <span
class="menulabel">File-&gt;Prepare Submission</span> menu item. You
will be prompted to save your entry and email it to the database you
selected. The address for GenBank is <tt>gb-sub@ncbi.nlm.nih.gov</tt>.
The address for EMBL is <tt>datasubs@ebi.ac.uk</tt>. The address for
DDBJ is <tt>ddbjsub@ddbj.nig.ac.jp</tt>.</p>

<a name="Advanced" id="Advanced"></a>
<h2>Advanced Topics</h2>

<a name="FeatureEditorDesign" id="FeatureEditorDesign"></a>
<h3>Feature Editor Design</h3>

<p>Sequin uses a common structure for all feature editor forms, with
(usually) three top-level folder tabs. One folder tab page is specific
to the given feature type (biological source and publications have
more). The <span class="folderlabel">Properties</span> and <span
class="folderlabel">Location</span> pages are common to all features.
Some of these pages may have subpages, accessed by a secondary set of
smaller folder tabs. This organization allows editors for complex data
structures to fit in a reasonably small window size. The most important
information in a given section is always presented in the first
subpage.</p>

<a name="CodingRegionPage" id="CodingRegionPage"></a>
<h4>Coding Region Page</h4>

<p><img class="figure" src="images/cds_edit.png" alt=
"Coding Region Page" /></p>

<p>The coding region editor is perhaps the most complicated form in
Sequin. Within the <span class="folderlabel">Coding Region</span> page,
the <span class="folderlabel">Product</span> subpage lets you predict
the coding region intervals from the protein sequence or translate the
protein sequence from the location. (Importing a protein sequence from
a file will also interpret the [gene=...] and [protein=...] definition
line information and automatically attempt to predict the coding region
intervals.) It also displays the genetic code used for translation and
the reading frame. (Please note that there are currently 17 different
genetic codes present in Sequin. For more information on these, see <a
href= "http://www.ncbi.nlm.nih.gov/Taxonomy/">
<tt>http://www.ncbi.nlm.nih.gov/Taxonomy/</tt></a>.)</p>

<p>The <span class="folderlabel">Protein</span> subpage lets you set
the name (or, if not known, a description) of the protein product. The
<span class="folderlabel">Exceptions</span> subpage allows you to
indicate translation exceptions to the normal genetic code, such as
insertion of selenocysteine, suppression of terminator codons by a
suppressor tRNA, or completion of a stop codon by poly-adenylation of
an mRNA.</p>

<p>Additional annotation on the protein product might include a leader
peptide, transmembrane regions, disulfide bonds, or binding sites.
These can be added after setting the <span class="popuplabel">Target
Sequence</span> popup on the sequence viewer to the desired protein
sequence. You can also launch a duplicate view, already targeted to the
appropriate protein, from the <span class="folderlabel">Protein</span>
subpage.</p>

<a name="PropertiesPage" id="PropertiesPage"></a>
<h4>Properties Page</h4>

<p><img class="figure" src="images/props_pg.png" alt=
"Properties Page" /></p>

<p>All features have a number of fields in common. The <span
class="buttonlabel">Partial</span> box will be checked if the 5'
partial or 3' partial boxes on the <span
class="folderlabel">Location</span> page were selected. <span
class="buttonlabel">Exception</span> means that the sequence of the
protein product doesn't match the translation of the DNA sequence
because of some known biological reason (e.g., RNA editing). The <span
class="popuplabel">Evidence</span> popup is now deprecated by the <span
class="folderlabel">Evidence</span> subpage.</p>

<p>In addition, nucleotide features (other than genes themselves)
can reference a gene feature. This is frequently done by overlap.
(The overlapping gene will show up on the feature as a /gene
qualifier in GenBank format.) Extension of the feature location
will automatically extend the gene that is selected in the editor.
In rare cases, you may want to set a gene by cross-reference.</p>

<p>The <span class="folderlabel">Comment</span> subpage allows text to
be associated with a feature. In GenBank format, this appears as a
/note qualifier. The <span class="folderlabel">Citations</span> subpage
attaches citations to the feature. (The citations should first be added
to the record using items in the <span
class="menulabel">Annotate-&gt;Publication</span> submenu, whereupon it
will appear in the REFERENCE section.) For example, an article that
justifies a non-obvious or controversial biological conclusion would be
cited here. In GenBank format, for example, if the publication is
listed as Reference 2, the feature citation appears as /citation=[2].
<span class="folderlabel">Cross-Refs</span> are cross-references to
other databases. The contents of this subpage may only be changed by
the GenBank, EMBL, or DDBJ database staff. <span
class="folderlabel">Evidence</span> has experiment and inference
qualifier fields. The experiment qualifier must include details of the
experiment used to justify the annotation.</p>

<a name="LocationPage" id="LocationPage"></a>
<h4>Location Page</h4>

<p><img class="figure" src="images/loc_page.png" alt=
"Location Page" /></p>

<p>All features are required to have a location, i.e., one or more
intervals on a sequence coordinate. The <span
class="folderlabel">Location</span> page provides a spreadsheet for
entering and editing this information. An arbitrary number of lines can
be entered. In this coding region example, the intervals correspond to
the exons. For an mRNA, the intervals would be the exons and UTRs. The
5' Partial and 3' Partial check boxes will show up as
&lt; or &gt; in front of a feature coordinate in the GenBank flatfile,
indicating partial locations.</p>

<p>The GenBank flatfile view of this location would be:</p>

<pre>
join(201..224,1550..1920,1986..2085,2317..2404,2466..2629)
</pre>

<p>If the <span class="buttonlabel">5' Partial</span> or <span
class="buttonlabel">3' Partial</span> boxes were checked, &lt; and &gt;
symbols would appear at the appropriate end of the join statement:</p>

<pre>
join(&lt;201..224,1550..1920,1986..2085,2317..2404,2466..&gt;2629)
</pre>

<p>If the sequence was reverse complemented (based on a length of 2881
nucleotides), the <span class="popuplabel">Strand</span> popups would
all indicate <span class="popuplabel">Minus</span>, and the join
statement for the resulting feature location would be as follows:</p>

<pre>
complement(join(253..416,478..565,797..896,962..1332, 2658..2681))
</pre>

<a name="NCBIDesktop" id="NCBIDesktop"></a>
<h3>NCBI DeskTop</h3>

<p><img class="figure" src="images/desktop.png" alt=
"NCBI DeskTop Window" /></p>

<p>The NCBI DeskTop is a window that directly displays the internal
structure of the record being viewed in Sequin. It can be
understood as a Venn diagram.</p>

<p>As with other views on a record, the DeskTop indicates selected
items and lets you select items by clicking.</p>

<p>In this example, Sequin was given the genomic nucleotide and protein
sequences for <span class="taxonomy">Drosophila melanogaster</span>
eukaryotic initiation factor 4E. It then determined the coding region
intervals and built an initial structure. The organism (BioSource
descriptor) is at the nuc-prot set and thus applies to both the
nucleotide and protein sequences.</p>

<a name="AdditionalInformation" id="AdditionalInformation"></a>
<h3>Additional Information</h3>

<p>The Sequin homepage <a href=
"http://www.ncbi.nlm.nih.gov/Sequin/">
<tt>http://www.ncbi.nlm.nih.gov/Sequin/</tt></a>
has a Frequently Asked Questions section and more detailed
instructions on using the capabilities of network-aware Sequin.</p>

<a name="Reference" id="Reference"></a>
<h2>Reference</h2>

<a name="NetworkConfiguration" id="NetworkConfiguration"></a>
<h3>Network Configuration</h3>

<p><img class="figure" src="images/net_cfg.png" alt=
"Network Configuration Form" /></p>

<p>When first downloaded, Sequin runs in stand-alone mode, without
access to the network. However, the program can also be configured
to exchange information with the NCBI (GenBank) over the Internet.
The network-aware mode of Sequin is identical to the stand-alone
mode, but it contains some additional useful options.</p>

<p>Sequin can only function in its network-aware mode if the
computer on which it resides has a direct Internet connection.
Electronic mail access to the Internet is insufficient. In general,
if you can install and use a WWW browser on your system, you should
be able to install and use network-aware Sequin. Check with your
system administrator or Internet provider if you are uncertain as
to whether you have direct Internet connectivity.</p>

<p>To launch the configuration form, select Net Configure under the
Misc menu, from either the initial Welcome to Sequin form or from a
viewer on an existing sequence record.</p>

<p>If you are not behind a firewall, set the <span
class="buttonlabel">Connection</span> control to <span
class="buttonlabel">Normal</span>. If you also have a Domain Name
Server (DNS) available, you can now simply press <span
class="buttonlabel">Accept</span>.</p>

<p>If DNS is not available, uncheck the <span
class="buttonlabel">Domain Name Server</span> box. If you are behind
a firewall, set the <span class="buttonlabel">Connection</span> control
to <span class="buttonlabel">Firewall</span>. The <span
class="buttonlabel">HTTP Proxy Server</span> box then becomes active.
If you also use a proxy server, type in its address. (If you have
access to DNS, it will be of the form
<tt>www.myproxy.myuniversity.edu</tt>. If you do not have DNS, you
should use the numerical IP address of the form <tt>127.45.23.6</tt>.)
Once you type something in the <span class="buttonlabel">HTTP Proxy
Server</span> box, the <span class="buttonlabel">Port</span> box
becomes active and can be filled in or changed as appropriate. (By
default the <span class="buttonlabel">Non-transparent Proxy
Server</span> box is empty, indicating a CERN-like proxy.) Ask your
network administrator for advice on the proper settings to use.</p>

<p>If you are in the United States, the default <span
class="textlabel">Timeout</span> of 30 seconds should suffice. From
foreign countries with poor Internet connection to the U.S., you can
select up to 5 minutes as the timeout.</p>

<p>Finally, you will need to quit and restart Sequin ifor the
network-aware settings to take effect.</p>

<p>If you are behind a firewall, it must be configured correctly to
access NCBI services. Your network administrator may have done this
already. If not, please have them contact NCBI for further
instructions on setting up firewalls to work with NCBI
services.</p>

<p><b>The following section is intended for network
administrators:</b></p>

<p>Using NCBI services from behind a security firewall requires
opening ports in your firewall. Please consult <a href=
"http://www.ncbi.nlm.nih.gov/IEB/ToolBox/NETWORK/firewall.html">
<tt>http://www.ncbi.nlm.nih.gov/IEB/ToolBox/NETWORK/firewall.html</tt></a>
for the list of current hosts and ports that have the firewall
daemon configured.</p>

<p>If your firewall is not transparent, the firewall port number
should be mapped to the same port number on the external host.</p>

<p>Note: Old NCBI clients used different application configuration
settings and ports than listed above. If you need to support such
clients, which are becoming obsolete, please contact <a href=
"mailto:info@ncbi.nlm.nih.gov"><tt>info@ncbi.nlm.nih.gov</tt></a>
for further information.</p>

<a name="FeatureTableFormat" id="FeatureTableFormat"></a>
<h3>Feature Table Format</h3>

<p>Sequin can now annotate features by reading in a tab-delimited
table. This is most often used by genome centers that store feature
interval information in relational databases or spreadsheets. For
most submitters, it is usually better to supply protein sequences
in FASTA format with gene and protein names embedded in the
definition line.</p>

<p>The feature table specifies the location and type of each feature,
and Sequin processes the feature intervals and translates any CDSs. The
table is read in the record viewer (after the sequence has been
imported) using the <span class="menulabel">File-&gt;Open</span> menu
item. The table must follow a defined format. The first line starts
with &gt;Feature, a space, and then the Sequence ID of the sequence you
are annotating. In the example below, eIF4E is the Sequence ID, and it
is a local identifier.</p>

<p>The table is composed of five columns: start, stop, feature key,
qualifier key, and qualifier value. The columns are separated by
tabs. The first row for any given feature has start, stop, and
feature key. Additional feature intervals just have start and stop.
The qualifiers follow on lines starting with three tabs.</p>

<p>For example, a table that looks like this:</p>

<pre>
&gt;Features lcl|eIF4E
80      2881    gene
                        gene     eIF4E

201     224     CDS
1550    1920
1986    2085
2317    2404
2466    2629
                        product  eukaryotic initiation factor 4E-II

1402    1458    CDS
1550    1920
1986    2085
2317    2404
2466    2629
                        product  eukaryotic initiation factor 4E-I
                        note     encoded by two messenger RNAs

80      224     mRNA
1550    1920
1986    2085
2317    2404
2466    2881
                        product  eukaryotic initiation factor 4E-II

80      224     mRNA
892     1458
1550    1920
1986    2085
2317    2404
2466    2881
                        product  eukaryotic initiation factor 4E-I

80      224     mRNA
1129    1458
1550    1920
1986    2085
2317    2404
2466    2881
                        product  eukaryotic initiation factor 4E-I
</pre>

<p>will result in a GenBank flatfile that contains this:</p>

<pre>
     mRNA            join(80..224,1129..1458,1550..1920,1986..2085,2317..2404,
                     2466..2881)
                     /gene="eIF4E"
                     /product="eukaryotic initiation factor 4E-I"
     mRNA            join(80..224,892..1458,1550..1920,1986..2085,2317..2404,
                     2466..2881)
                     /gene="eIF4E"
                     /product="eukaryotic initiation factor 4E-I"
     mRNA            join(80..224,1550..1920,1986..2085,2317..2404,2466..2881)
                     /gene="eIF4E"
                     /product="eukaryotic initiation factor 4E-II"
     gene            80..2881
                     /gene="eIF4E"
     CDS             join(201..224,1550..1920,1986..2085,2317..2404,2466..2629)
                     /gene="eIF4E"
                     /codon_start=1
                     /product="eukaryotic initiation factor 4E-II"
                     /translation="MVVLETEKTSAPSTEQGRPEPPTSAAAPAEAKDVKPKEDPQETG
                     EPAGNTATTTAPAGDDAVRTEHLYKHPLMNVWTLWYLENDRSKSWEDMQNEITSFDTV
                     EDFWSLYNHIKPPSEIKLGSDYSLFKKNIRPMWEDAANKQGGRWVITLNKSSKTDLDN
                     LWLDVLLCLIGEAFDHSDQICGAVINIRGKSNKISIWTADGNNEEAALEIGHKLRDAL
                     RLGRNNSLQYQLHKDTMVKQGSNVKSIYTL"
     CDS             join(1402..1458,1550..1920,1986..2085,2317..2404,
                     2466..2629)
                     /gene="eIF4E"
                     /note="encoded by two messenger RNAs"
                     /codon_start=1
                     /product="eukaryotic initiation factor 4E-I"
                     /translation="MQSDFHRMKNFANPKSMFKTSAPSTEQGRPEPPTSAAAPAEAKD
                     VKPKEDPQETGEPAGNTATTTAPAGDDAVRTEHLYKHPLMNVWTLWYLENDRSKSWED
                     MQNEITSFDTVEDFWSLYNHIKPPSEIKLGSDYSLFKKNIRPMWEDAANKQGGRWVIT
                     LNKSSKTDLDNLWLDVLLCLIGEAFDHSDQICGAVINIRGKSNKISIWTADGNNEEAA
                     LEIGHKLRDALRLGRNNSLQYQLHKDTMVKQGSNVKSIYTL"
</pre>

<p>Note that if the gene feature spans the intervals of the CDS and
mRNA features for that gene, you don't need to include gene
"qualifiers" in those features, because they will be picked up by
overlap.</p>

<p>Features that are on the complementary strand are indicated by
reversing the interval locations. For example, the table:</p>

<pre>
&gt;Features lcl|dna2
5284    5202    tRNA
                        product  tRNA-Glu
</pre>

<p>will result in a GenBank flatfile containing:</p>

<pre>
     tRNA            complement(5202..5284)
                     /product="tRNA-Glu"
</pre>

<p>More instructions on using the feature table format for
submitting large genomic records are available at<br />
<a href="http://www.ncbi.nlm.nih.gov/Sequin/table.html">
<tt>http://www.ncbi.nlm.nih.gov/Sequin/table.html</tt></a>.</p>

<hr />

<div id="footer">
<b>Questions or Comments?</b><br />
Write to the <a href="mailto:info@ncbi.nlm.nih.gov">NCBI Service Desk</a>
<br />
<br />
Revised August 21, 2007<br />
</div>

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