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<h1 id="submitting-mitochondrial-and-chl">Submitting Mitochondrial and Chloroplast Genomes to GenBank</h1>
<h1 id="introduction">Introduction</h1>
<p>This document guides first timers through the entire process of
submitting organelle genome(s) to GenBank. It also provides help for the
submitters who were asked to resubmit their data because of lacking or
inaccurate annotation. If you are already familiar with the submission
process but you need to fix your annotation, go to the following
sections of the document:</p>
<p><a href="#path">Organelle Genome Submission Path</a></p>
<p><a href="#Bankit">Working with Bankit</a></p>
<p><a href="#Features">The Features Step/Annotation with the Five-Column Feature Table</a></p>
<p><a href="#trouble">Troubleshooting Annotation/Feature Table Format</a></p>
<h1 id="path">Organelle Genome Submission Path</h1>
<p>Use BankIt to submit complete or incomplete organelle genome(s) to
GenBank:</p>
<ol>
<li>
<p>On the <a href="https://www.ncbi.nlm.nih.gov/WebSub/">BankIt web page</a>
select the following radio button:</p>
</li>
<li>
<p><em>Sequence data not listed above (through BankIt): mRNA, genomic DNA,
organelle, ncRNA, plasmids, other viruses, phages, synthetic
constructs</em></p>
</li>
<li>
<p>Click the <strong>Start</strong> button (sign in to NCBI if needed).</p>
</li>
<li>
<p>On the resulting page, select the <strong>Start BankIt Submission</strong>
button.</p>
</li>
</ol>
<p>You can submit more than one organelle genome with one BankIt. However,
you may need to separate your data into several BankIt submissions if you have
numerous and/or large genomes. We cannot provide a hard limit for the
data size of one BankIt as it will depend on your connection speed. If
you get a server error while working in BankIt, your data size may be
too large. You can use the
<a href="https://www.ncbi.nlm.nih.gov/genbank/tbl2asn2/">tbl2asn</a> command line
software as the alternative tool for large data. You would use the same
data input files (same formats) as in BankIt.</p>
<p>GenBank does not require reads from next generation sequencing
technologies to accompany the assembled organelle genome submissions.
However, you can submit your reads to <a href="https://www.ncbi.nlm.nih.gov/sra/">Sequence Reads Archive
(SRA)</a> if that is necessary for your project.
Please refer to the <a href="https://www.ncbi.nlm.nih.gov/sra/docs/submit/">SRA submission
guide</a>. Once your SRA and
GenBank records are processed, GenBank indexers can link your GenBank
accessions with your SRA data.</p>
<p>Submit organelle genomes through the <a href="https://submit.ncbi.nlm.nih.gov/subs/genome/">Genome Submission
Portal</a> ONLY if they
accompany organism's nuclear genome submission.</p>
<h1 id="Bankit">Working with BankIt</h1>
<p>BankIt is a web-based application comprised of several steps that
include: <strong>Contact</strong>, <strong>Reference</strong>, <strong>Sequencing Technology</strong>,
<strong>Nucleotide</strong>, <strong>Submission Category</strong>, <strong>Source Modifiers</strong>,
<strong>Features</strong>, and <strong>Review and Correct</strong>. You will find comprehensive
instructions within BankIt at each step. Here, we selectively provide
instructions for three of the steps: <strong>Nucleotide</strong>, <strong>Source
Modifiers</strong>, and <strong>Features</strong>. We are emphasizing parts that are
relevant to organelle genome submissions.</p>
<h2 id="uploading-genome-sequence-at-the">Uploading genome sequence at the Nucleotide step</h2>
<p>The Nucleotide page contains a section where you mark your genome(s) as
complete, circular genomic DNA:</p>
<p><img src="/core/assets/genbank/images/org_submit_1.png" alt="org submit 1" height="200px" width="750px" /></p>
<p>On the same page, you will upload your sequence(s) in the FASTA format
(not aligned) or in an alignment format such as FASTA+GAP or NEXUS. All
the formats are described/explained at the step in BankIt. Here, we
illustrate a plain text file that contains three genomes (the sequences
are truncated for illustration purpose):</p>
<p><img src="/core/assets/genbank/images/org_submit_2.png" alt="org submit 2" /></p>
<p>Each FASTA starts with the "<strong>&gt;</strong>"symbol followed by a sequence
identifier (Sequence_ID). You can choose your own Sequence_IDs but
keep them simple. Inclusion of the organism name within the square
brackets allows for proper processing of the name from the FASTA
definition line. You can follow the organism name by a descriptive text.
It is important that you present the entire FASTA definition in a single
line and then follow by a hard return before the sequence.</p>
<p>GenBank indexers will edit the definition lines in the published records
according to GenBank standards, so you should not be overly concerned
about matching your definition line to those in the database.</p>
<h2 id="describing-sequence-source-at-th">Describing sequence source at the Source Modifiers Step</h2>
<p>The Source Modifiers page contains the <strong>Organelle/Location</strong> drop-down
menu where you mark the organelle genome that you have:</p>
<p><img src="/core/assets/genbank/images/sourcea.png" alt="org submit 3" /></p>
<h2 id="providing-source-modifiers-using">Providing source modifiers using the web form</h2>
<p>Choose applicable source modifiers that allow you to describe your
sample from the <strong>source modifier</strong> pull down menu. Provide values as
you have them for your sample. Commonly applicable source modifiers for
organelle genomes are <strong><em>strain/isolate and/or voucher</em></strong>, <strong><em>geo_loc_name,
and isolation_source</em></strong>. Here, we illustrate an example with detailed
source information for the mitochondrial genome of Chinese short-limbed
skink. The left hand-side of the image below (blue background) shows the
selected modifiers and values as the submitter would have provided in
BankIt. The right hand-side (yellow background) reflects the information
in the <strong>source</strong> section of the processed <a href="https://www.ncbi.nlm.nih.gov/nuccore/MW327509.1">GenBank
record</a>:</p>
<p><img src="/core/assets/genbank/images/org_submit_4a.png" alt="org submit 4" height="500px" width="850px" /></p>
<p>Note that GenBank indexers (not the submitter) provide some parts of
source information (such as the Taxonomy <strong>db_xref</strong> ).</p>
<h2 id="providing-source-modifiers-using_1">Providing source modifiers using a table (file input)</h2>
<p>If you are submitting more than one genome and you need to provide
different source values for each genome, you need to provide a source
table (file input). The web form will not work in such case.</p>
<p>Provide <strong><em>unique identifiers</em></strong> to differentiate between the samples of
the same species. For example, use <strong><em>isolate</em></strong> and provide the
alphanumerical codes that you used in your study to differentiate
between your samples. Other source modifiers that can serve as
unique identifiers include <strong><em>strain</em></strong>, <strong><em>clone</em></strong>,
<strong><em>culture_collection</em></strong>, and <strong><em>specimen_voucher</em></strong>.</p>
<p>The source table for our example of the three Physalis chloroplast
genomes shows unique isolate designations, different collection dates,
and the same geographic location name/region. Note the first column of the table
contains Sequence_IDs that match those in the FASTA file.</p>
<p>To format a source table, you can work with a spreadsheet:</p>
<p><img src="/core/assets/genbank/images/org_submit_5a.png" alt="org submit 5" /></p>
<p>Once completed, save the table as plain-text, tab delimited file (you
cannot upload a spreadsheet directly in BankIt):</p>
<p><img src="/core/assets/genbank/images/org_submit_6a.png" alt="org submit 6" /></p>
<p>Before uploading the file in BankIt, you should also check for any
discrepancies in your data (especially the collection_date) that the
spreadsheet software might have automatically introduced.</p>
<p>INSDC has approved a list of <a href="https://www.insdc.org/technical-specifications/missing-value-reporting/">null terms</a> that should be used for reporting missing values in /collection_date and /geo_loc_name.</p>
<h1 id="Features">The Features Step/Annotation with the Five-Column Feature Table</h1>
<p>The <strong>Features</strong> step is an essential step in submitting your organelle
genome(s). You are required to provide genes, coding regions (CDS), and
other feature annotations on your genomes. Submitting your sequences
without annotation or inaccurate annotation will delay issuing your
accession numbers. GenBank indexers will ask you to resubmit your
genomes with added/corrected annotation. It means that you will need to
start a new BankIt submission.</p>
<p>BankIt offers two ways of providing features: (1) by completing input
forms and (2) by uploading a five-column feature table. Input forms are
impractical for annotating organelle genomes with multiple features as
you would need to work through the forms for each individual feature.
Hence, the five-column feature table is currently your only option to
provide annotation for organelle genomes.</p>
<p>The format of the feature table is explained in BankIt at the
<strong>Features</strong> step. Our illustrated example shows a small section of the
feature table as the submitter of the Chinese short-limbed skink
mitochondrion (the
<a href="https://www.ncbi.nlm.nih.gov/nuccore/MW327509.1">MW327509.1</a> record)
would have prepared:</p>
<p><img src="/core/assets/genbank/images/org_submit_7.png" alt="org submit 7" height="500px" width="850px" /></p>
<p>The top panel (blue background) shows how to format the table using
spreadsheet software. Just like with the source table, you need to save
your final feature table as a plain-text tab-delimited file before you
can import it in BankIt. If you are working directly in a text editor,
you need to move from one column to another with a single stroke on the
<strong>tab</strong> key on your computer keyboard.</p>
<p>The total number of columns in the feature table is five. For each
feature, locations are provided in columns 1 and 2. For a feature on the
complement strand, invert the locations (the larger one of the two
should be in column 1). The feature key is in column 3. Qualifiers and
their values that follow are offset in columns 4 and 5.</p>
<p>The bottom panel (yellow background) in the image reflects the
information in the <strong>FEATURES</strong> section of the processed <a href="https://www.ncbi.nlm.nih.gov/nuccore/MW327509.1">GenBank
record</a>. The processed
record has additional qualifiers that were not in the original feature
table. BankIt will use codon_start 1 (reading frame 1) as default if
none is provided in the feature table.</p>
<p>You should not be providing protein_ids in a feature table as these
will be uniquely assigned for each translated CDS on your genome at the
time your record is processed.</p>
<p>The feature file that you import in BankIt should contain the features
of all the sequences that you imported at the Nucleotide step. For
example, there will be three sets of feature annotations in the file for
the three Physalis chloroplast genomes. Each starts with a feature
definition that contains the "&gt;" symbol and the word "Feature". The
word "Feature" is followed by a space and then by the sequence ID that
matches in the corresponding FASTA sequence:</p>
<p><img src="/core/assets/genbank/images/org_submit_8.png" alt="org submit 8" /></p>
<p>You should always provide the feature locations as they are on the
genome that you are submitting. You are required to determine the
correct feature locations.</p>
<p>Your annotation software may offer the five-column feature table as one
of the annotation outputs. However, as we detail in the troubleshooting
section, you need to review the annotation and the table format to meet
<a href="http://www.insdc.org/documents/feature_table.html">GenBank (INSDC)
standards</a>.</p>
<p>You can also use a feature table from a GenBank record that represents a
similar genome to the one that you are submitting as your template. The
troubleshooting section provides steps for obtaining a template from the
NCBI web and a tip for removing unwanted information from the table.</p>
<p>Once you have prepared the table, upload it in BankIt at the
<strong>Features</strong> step, by selecting the following button:</p>
<ul>
<li><strong><em>Add features by uploading five column feature table file</em></strong></li>
</ul>
<p>Uploading the file is a two-step process. First use the <strong>Choose File</strong>
button, then the <strong>Upload file</strong> button. Upon uploading the table,
BankIt will translate the coding regions based on their annotation
(locations). If you are working with a known organism, BankIt will
recognize the organism and apply the proper <a href="https://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=cgencodes">translation table
(genetic
code)</a>
for translation. However, if the organism is not yet in the database
(unknown), you need to provide the genetic code for each CDS in the
table by introducing the transl_table qualifier as we show in our
illustrated examples above.</p>
<p>BankIt will also validate your submission. Address any error reports,
such as internal stop codons in protein translation. Use other
approaches as listed in the troubleshooting section to assure accuracy
of your annotation.</p>
<p>Once you complete your submission, GenBank indexers will communicate
with you to address any remaining issues and assign accession numbers
for your records. Your records will require manual processing before
they are publicly released.</p>
<p>Do not send a new submission if you cannot find your records in GenBank.
Please write <a href="mailto:gb-admin@ncbi.nlm.nih.gov">gb-admin@ncbi.nlm.nih.gov</a> and inquire about the status.</p>
<h1 id="trouble">Troubleshooting Annotation/Feature Table Format</h1>
<p>Annotation programs and copying features from a similar genome is a good
way to begin annotating a new genome. However, you need to review the
resulting annotations for accuracy and completeness. For example,
annotation programs may put on a partial coding region because it cannot
determine the complete coding region. Or an essential gene may be annotated
as a <strong>misc_feature</strong> because there is an internal stop codon in
translation.</p>
<h2 id="if-using-an-annotation-program">If using an annotation program:</h2>
<ol>
<li>
<p>Determine that the complete coding region has been annotated. Use
other tools such as BLAST to evaluate the accuracy of the predicted
starts and stops of the coding regions. If several features are
partial this indicates that the annotation program was unable to
determine the correct endpoints.</p>
</li>
<li>
<p>Review the <strong>product</strong> names. The following are examples of product
names that you should modify/change:</p>
<p>a. beginning with 'TPA:'</p>
<p>b. containing the phrase 'No product string in profile'</p>
<p>c. containing the word 'partial' or a bracketed term</p>
</li>
<li>
<p>Review the <strong>gene</strong> features to assure that they have corresponding
CDS/tRNA/rRNA features. A <strong>gene</strong> feature should not be
listed or linked with a <strong>misc_feature</strong>. Lacking CDS features for
protein-coding genes usually indicate the annotation software cannot
predict a valid conceptual translation due to presence of internal
stop codons or missing the start and/or stop codon.</p>
</li>
<li>
<p>You should check your sequence/assembly quality if the annotation
tools are failing with CDS annotation of essential genes. You can
find guidance on using BLAST for checking sequence quality in a
<a href="https://support.nlm.nih.gov/knowledgebase/article/KA-05223/en-us">series of knowledgebase
articles</a>.</p>
</li>
<li>
<p>Annotation tools may introduce unwanted annotation lines/features
that do not meet INSDC standards. Remove the lines/features that
contain:</p>
<p>a. <strong>misc_features</strong> with the annotation program name.</p>
<p>b. the '<strong>label</strong>' term.</p>
<p>c. systematic locus tags such as 'gene 1'.</p>
<p>d. the <strong>standard_name</strong> qualifier</p>
<p>e. BLAT output</p>
</li>
</ol>
<h2 id="if-copying-features-from-a-simil">If copying features from a similar genome:</h2>
<ol>
<li>
<p>Exclude (remove): <strong>locus_tags</strong> (these must be unique for each
genome if included), <strong>gene_xref</strong>, <strong>GeneID</strong>, and <strong>protein_ID</strong>
lines from the table.</p>
</li>
<li>
<p>Check that the annotation is reasonable. For example, a variation
feature on one genome may not apply to your genome.</p>
</li>
</ol>
<h2 id="downloading-and-adjusting-featur">Downloading and adjusting feature table template:</h2>
<p>You might have identified a GenBank record that can serve as an
annotation template. Here we use the
<a href="https://www.ncbi.nlm.nih.gov/nuccore/MT161478">MT161478.1</a> record as an
example. To download its feature table:</p>
<ul>
<li>
<p>Click the <strong>Send to</strong> link top right above the record.</p>
</li>
<li>
<p>Select: <strong>Complete record</strong> <strong>File</strong> <strong>Feature table</strong></p>
</li>
<li>
<p>Click the <strong>Create File</strong> button</p>
</li>
</ul>
<p>The downloaded file will contain <strong>protein_id</strong> qualifiers (lines) that
you need to remove.</p>
<p>You can use various text editors functions to remove repetitive unwanted
lines. For example, open the downloaded file with the <a href="https://notepad-plus-plus.org/">Notepad++
editor</a>. To remove the lines:</p>
<ul>
<li>
<p>Select: <strong>Search</strong> <strong>Mark...</strong></p>
</li>
<li>
<p>Check the <strong>Bookmark line</strong> check box in the <strong>Mark</strong> dialogue.</p>
</li>
<li>
<p>Enter: <em>protein_id</em> as your search term in the <strong>Find what</strong> box of
the <strong>Mark</strong> dialogue.</p>
</li>
<li>
<p>Click the <strong>Mark All</strong> button.</p>
</li>
<li>
<p>Close the <strong>Mark</strong> dialogue.</p>
</li>
<li>
<p>Select: <strong>Search</strong> <strong>Bookmark</strong> <strong>Remove Bookmarked lines</strong></p>
</li>
</ul>
<h2 id="annotation-checks-for-individual">Annotation Checks for individual organism groups:</h2>
<p>Here are some of the basic annotation checks that you
need to meet for your organism groups:</p>
<p>I. Metazoan Mitochondria</p>
<ul>
<li>Each genome should have 13 CDS features, 22 tRNAs and 2 rRNAs.</li>
</ul>
<blockquote>
<p>There are known exceptions such as Cnidaria and Porifera. If your annotation is missing one of the expected features, provide an explanation. You can add a message for the indexers on the last BankIt page, <strong>Review and Correct</strong>.</p>
</blockquote>
<ul>
<li>
<p>The CDS feature rarely overlaps the tRNA feature by more than a few nucleotides. These coding regions use the mitochondrial TAA stop.</p>
</li>
<li>
<p>Use protein BLAST to evaluate the length and similarity of the predicted proteins against those from related organisms.</p>
</li>
<li>
<p>With few exceptions (for example mollusks), the features are on both strands. Check the strandedness of the tRNAS and rRNAs.</p>
</li>
</ul>
<p>II. Fungi</p>
<ul>
<li>Intronic ORFs should be located within an intron.</li>
</ul>
<p>III. Embryophyta Plants</p>
<ul>
<li>
<p>Rps12 is a trans-spliced gene consisting of 2-3 exons. This gene is not RNA edited.</p>
</li>
<li>
<p>Check the size of the tRNAs. tRNAs can consist of two exons in some cases.</p>
</li>
<li>
<p>Commonly RNA edited genes are ndhD, psbL, rpl2.</p>
</li>
<li>
<p>The ribosomal RNAs that should be annotated: 4.5S, 5S, 16S and 23S ribosomal RNA.</p>
</li>
</ul>
<p>For more help, contact the NCBI helpdesk at: <a href="mailto:info@ncbi.nlm.nih.gov">info@ncbi.nlm.nih.gov</a> .</p>
<p>If you need to update already accessioned records, follow the
instructions on the <a href="https://www.ncbi.nlm.nih.gov/genbank/update/">GenBank Update
page</a>.</p>
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