BOLD Handbook.4765687567354634534653457346534

BARCODE OF LI FE DATA SYSTEMS
Handbook
October 2008
BOLDSYSTEMS.org
B A R C O D E O F L I F E D A T A S Y S T E M S
BOLDSYSTEMS.org BBO BO BO BO BO BO BB LD LD LD LDD LDSY SY SY SSSSY SY SYST ST ST ST ST STEM EM EM EMMMM EM EMS. S. S. S. S. SSS oor or or rggggggg BOLDSYSTEMS.org
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Tabl e of Contents
1. I ntroducti on
1. Introduction
2. BOLD General System Map
3. Signing up for BOLD
4. Taxonomy Browser
5. BOLD Search
6. Create a BOLD Project
7. Submission Protocols
a) Data Submission
b) Image Submission
c) Trace Submission
d) Sequence Submission
8. BOLD Project Summary
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The Barcode of Life Data System (BOLD) is an informatics workbench aiding the acquisition, storage, analysis and
publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a
traditional bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By
providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE
designation in the global sequence databases. Because of its web-based delivery and exible data security model,
it is also well positioned to support projects that involve broad research alliances.
This handbook provides details on how to sign up for BOLD and create a project. It also explains how to upload
specimen data, images, traces and sequences to your project on BOLD.
Figure 1-1: The front page of BOLD.
BOLDSYSTEMS.org
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2. BOLD General System Map
Project Management from Project Console or Record Listing Page
BOLDSYSTEMS
www.barcodinglife.org
Tax Browser
ID Specimen
Private
Projects*
(log-in)
Published
Projects*
Documentation
Uploads
Specimen Data
Images
Traces
Sequences
Primers
View All
Primers
Register
Primers
Browse
Hierarchy
Download
Sequences
Taxon
Search
Taxon
ID Tree
Request
an account
Search
All
Records
BOLD
BOLD
Tutorial
Manuals Templates
Trace Image Data Trace Image Data
ITS Database Identification COI Database Identification
Species
Level
Specimen ID
All Barcodes
Specimen ID
Reference
Specimen ID
Specimen
ID
Species
Page
These functions are only available from the private project console
*The published projects are
also accessible when a user
is signed in to the private
projects workspace
Legend
Manual
Input
Viewable
Data
Downloadable
Data
Action Document Analysis
Create
New
Project
Species
Barcoded
Report
Publication
Project
Summary
Submit to
Genbank
Downloads
Data
Spreadsheets
Traces
Sequences
Specimen Labels
Specimen Aggregates
Image
Library
Distribution
Map
Sequence Analysis
Taxon ID Tree
Distance
Summary
Sequence
Composition
Nearest
Neighbour
Spec Age vs
Seq Length
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3. Si gni ng up for BOLD
Getting an account on BOLD allows you to upload your
data into a private workspace and take advantage of the
integrated analytical tools.
On the BOLD main page (www.boldsystems.org) click
on either one of the two links: Request an Account or
Request a new user account. These links will take you to
the New User Application Form.
(http://www.boldsystems.org/views/newuserapp.php)
Click on Submit Request to send your application to
BOLD. An introductory e-mail will be sent to you with the
information you need to log in and begin using BOLD.
Once you have an account you can login via the main page
to access your private workspace. Your next step will be to
create a project. Please see page 4 for instructions.
Table 3-1: Information required to create a new user
account on BOLD.
Valid Email Address Use a current institutional email.
First Name Fill in your rst name, rst letter should
be capitalized
Middle Initial Fill in middle initial(s) if needed,
capitalized
Last Name Fill in your last name, rst letter should
be capitalized
Institutional
Afliation
Select the name of your institution
Add New
Institution
If your institution is not listed, click on
button to register it
Password Should be at least 5 characters
Figure 3-1: New user account creation on BOLD.
BOLDSYSTEMS.org
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4. BOLD Taxonomy Browser
The taxonomy browser allows
users to examine the progress
of DNA barcoding, and to
browse different levels of the
taxonomic hierarchy. Animals,
Plants, Fungi, and Protists are
being barcoded and the user
can browse through each
kingdom from phylum down
to the species level.
Table 4-1: Information available at each taxonomic level within the BOLD taxonomy browser.
Lineage Lists the higher taxonomic levels.
Specimen Records The number of specimen records.
Specimens with
Barcodes
The number of barcoded specimens.
Public Sequences The number of public sequences and a
link to download them.
List of Species
Barcoded
A list of all species with records on
BOLD. The number of specimens, the
number of sequences and the number of
sequences greater than 500bp are listed.
Link Outs Links to several community partners
pages for that specimen
Lower Taxonomy Links to all lower classications
Graphic Displays
of:
the total number of barcodes and
reference barcodes.
quantity of species barcodes and those
used as reference barcodes.
the institutions where the specimens
are deposited.
a map of the world highlighting specimen
collection locations.
a graph showing the frequency of
specimens/barcodes against age.
a list of countries where specimens
were collected, including the number of
specimens from each country.
various images of specimens within that
taxonomic group.
Figure 4-1: The BOLD taxonomy browser.
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5. BOLD Search
On the BOLD project list page, select the Search All Records
link on the top left hand side. There are two types of searches for
BOLD: Basic Search and Advanced Search.
Note that any search criteria containing spaces such as Species
names, country names that consist of more than one word, and
sample IDs with spaces should be wrapped in double quotes (eg
United States or Drosophila melanogaster). The Paste from
Spreadsheet function allows you to paste a column of sample IDs
or process IDs from a spreadsheet and will automatically place
quotes around search criteria that require them.
Table 5-2: Explanation of the terms used within the
Advanced BOLD search functions.
Taxonomy Searches the taxonomic names on BOLD.
There is a text eld for search terms that
should be either included or excluded
from the search
Geography -
Country/Province
Searches the country and province
names on BOLD. There is text eld
for search terms that should be either
included or excluded from the search
Geography -
Region
Searches region names on BOLD. A
text eld for search terms that should be
included in the search.
Sequence Length Text elds for each of the minimum and
maximum number of base pairs.
Specimen/Sample
ID
Searches sample IDs and process IDs on
BOLD. There is also the option of pasting
a list of sample or process IDs from a
spreadsheet (link to the right).
Include GenBank
Data
When checked, the search includes
GenBank records on BOLD.
Single
Representative
per Species
When checked, the search will only display
one representative per species found.
Table 5-1: Explanation of the terms used within the
Basic BOLD search functions.
Taxonomy Searches the taxonomic names on
BOLD. More information about the drill-
down menus and how they work would
go here, pending content from Megan.
Geography Country/FAO and State/Province:
Searches the country and province
names on BOLD
Figure 5-1: The BOLD search engine, showing both basic and advanced search functions.
BOLDSYSTEMS.org
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6. Creati ng a new proj ect
Once logged into BOLD, select the Create New Project link on the top left hand side of the project list
page. It will take you to the New Project Submission Form. The following pieces of information need to be
entered in order to create the project:
Sequence Access permissions consist of three levels. With Analyze permission, the user can perform analysis on the
data, but cannot view more than a summary of the data (sequence and related information remain hidden). With View
permission, the user can view or download the sequence data. With Edit permission, the user can upload sequences
or make changes to existing sequence features.
Specimen Access permission allows the user control over sample identiers, taxonomy, collection data, and images of
the specimen: this level is intended for project managers, collectors, and taxonomists only.
To submit your entries to BOLD, click Save at the bottom of the form.
Please note that the person who creates a project is automatically the project manager of that project. The
project manager has full access to the project and can assign other users to the project.
The project manager can change any details or add/remove users, by simply clicking on Modify Project Prop-
erties in the upper left corner of the project.
Table 6-1: Required information for BOLD project
creation.
Project Title Please create a descriptive name
Project Code A 3-5 letter code. It needs to be unique
across BOLD
Project Type Choose between the following options:
Data Project (contains specimen &
sequence records)
Folder Project (contains other
projects)
Primary Marker Select your primary marker. CO1 is the
default.
Cytochrome Oxidase Subunit 1 5
Region Interspacer (ITS) Region
Campaign Select the name of the campaign the
project is part of or None (General
Project) if it is not part of a campaign.
Place in
container
Select the name of the Folder Project or
Independent Project if it does not belong
into a folder project.
Project
Description
A brief summary of the use and intention
of the project.
Project Manager The person who creates a project is
automatically the project manager, and has
full specimen and sequence access.
Assign Users Other BOLD users can be added to a
project. Different levels of access are
possible:
Sequence Access: Analyze, View, and
Edit Sequences
Specimen Access: Edit Specimens
Figure 6-1: The BOLD new project submission form.
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Full Taxonomy Full taxonomy consisting of phylum*,
class, order, family, subfamily (optional),
genus, species binomial.
Identier Full name of primary individual respon-
sible for providing taxonomic identi-
cation of the specimen.
Identier E-mail E-mail address of the primary identier.
Identier Institution Institution of the identier.
Table 7a-2: Field denitions for Taxonomy page on
accompanying spreadsheet.
Sex Male/female/hermaphrodite only.
Reproduction Sexual/asexual/cyclic parthenogen only.
Life Stage Adult/immature only.
Extra Info User Specied Characteristics (free text) -
Can be displayed on a tree or used to sort
records. Limited to a maximum of 50 charac-
ters. Designate FAO region here.
Notes Free text or XML tagged text. All XML text
should be surrounded by the XML start
(<xml>) and stop (<xml>) tags.
Table 7a-3: Field denitions for Specimen Details page
on accompanying spreadsheet.
7a) Data Submi ssi on Protocol
This protocol assists in the submission of bulk data to BOLD. This is the easiest way to populate your project with records,
as well as the only way to enter new species taxonomy into the BOLD library. Described below is the necessary format of
the data that is required for a correct submission.
Whenever a bulk submission is sent to the data manager(boldsyst@uoguelph.ca), the following pieces of information need
to be sent in the body of the emai, with the standard submission spreadsheet attached:
Project title I.
Project code II.
Project manager III.
Priority Level (High, Intermediate or Low) IV.
Submission type (New Records or Update)* V.
* If type is Update: Please specify which worksheets (Voucher Info, Taxonomy, Specimen Details, or Collection Data) need to be up-
dated. See page 7 for more information.
The data spreadsheet consists of 4 worksheets, a main specimen identier worksheet (voucher info) that is linked to three
other worksheets: taxonomy, specimen details, and collection data. (Refer to Tables 1 through 4 for eld denitions)
Table 7a-1: Field denitions for Voucher info page on
accompanying spreadsheed.
Sampple ID * ID associated with the sampl p e being g
sequenced (often an extension of eld
or Museum ID).
Fi Fiel eldd ID ID ** Sp Spec ecim imen en iide dent nti ieerr fr from om aa ppri riva vate te
coll llec i tion or Fi Fi l eldd nu b mber ffrom a
collection event.
Mu Muse seum um IIDD * Ca Cata talo logg nu numb mber er iinn cu cura rate tedd co coll llec ecti tion on
for a vouchered specimen.
Collection Code Code associated with given collection.
Institution Storing * Full name of the institution where
sp spec ecim imen en iiss vo vouc uche here redd.
Sample Donor Full name of individual responsible for
providing specimen or tissue sample.
Donor E-mail E-mail of the sample donor.
* Minimum required elds for new records.
Table 7a-4: Field denitions for Collection Data page
on accompanying spreadsheet.
Collectors Comma delimited list of collectors.
Collection Date Date of collection, must be in MM-DD-
YYYY format.
Continent/Ocean ISO Continents and Oceans.
Country ISO Countries.
State/Province States and provinces (according to Getty
Geographical Thesaurus).
Region Park, county, district, lake or river.
Sector Sector of park or county/city.
Exact Site Description of collection location.
GPS Coordinates Latitude & Longitude in degrees.decimal
degrees format (e.g. 45.837).
Elevation/Depth Elevation or depth in meters.
BOLDSYSTEMS.org
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Data Submi ssi on - Exampl es
Here is an example of a properly lled in data submission. You can get this blank template in two ways:
From the info CD that came along with your sampling units from the CCDB
Online by clicking on Specimen Data under the Uploads menu the sheet is available through the link at the
top
Use the tabs at the bottom of the workbook to navigate through the four pages.
All of the data in BOLD is organized by projects. There is a limit of 1000 entries for a given project, to keep the size
manageable. Related projects can be grouped into containers. An individual entry in the database represents a barcode
of a given specimen. The Process ID uniquely represents a specimen in BOLD. This is the identier that is used to track
a specimen through the barcoding process: collection, taxonomic identication, sequencing, analysis and nal publication
of data. Process ID is assigned internally when a specimen record is created.
Specimen data can be entered in one of two ways. As outlined here, for larger sets of samples, the data can be entered
on the Data Submission Template spreadsheet and sent to BOLD. Data managers will review the data, to ensure that it
meets the minimum requirements, and input it to BOLD. For smaller numbers of entries, (ie: 1-10 records) users can
enter sample data through the web interface by clicking on Specimen Data under the Uploads menu and using the
manual interface there.
Specimen Info
Sample ID Field ID Museum voucher ID Collection Code Institution Storing Sample Donor Donor Email
Sample-demo01 Sample-demo01 BIO Joe Smith jsmith@BIO.org
Sample-demo02 Sample-demo02 15466-JUC-ISC ISC ROM Joe Smith jsmith@BIO.org
Sample-demo03 Sample-demo03 BIO Joe Smith jsmith@BIO.org
Figure 7a-1: Example data for Specimen Info
Taxonomy
Sample ID Phylum Class Order Family Subfamily Genus Species Identier Identier Email
Identier
Institution
Sample-demo01 Arthropoda Insecta Diptera Asilidae
Hydro-
psychinae
Efferia
Efferia
aestuans
Joe Smith jsmith@BIO.org Oxford
Sample-demo02 Arthropoda Insecta Diptera Asilidae Asilus Joe Smith jsmith@BIO.org Oxford
Sample-demo03 Arthropoda Insecta Diptera Joe Smith jsmith@BIO.org Oxford
Figure 7a-2: Example data for Taxonomy
Specimen Details
Sample ID Sex Reproduction Life Stage Extra Info Notes
Sample-demo01 Female Sexual Adult Commonly called Robber Fly
Sample-demo02 Male Sexual Adult feeding on fruit
Sample-demo03 Male Sexual Adult
Figure 7a-3: Example data for Specimen Details
Collection Info
Sample ID Collectors
Collection
Date
Continent
/ Ocean
Country
State /
Province
Region Sector
Exact
Site
Lati-
tude
Longi-
tude
Elevation
Sample-demo01 Joe Smith 27-Jul-07 Asia Japan Hokkaido
Izarigawa,
Eniwa
42.878 141.572 45
Sample-demo02 Joe Smith 27-Jul-07 Japan Hokkaido Soya 44.671 142.788
Sample-demo03 Joe Smith 5-Sept-07
Central
America
Costa
Rica
Guana-
caste
ACG
Mundo
Neuvo
10.772 -85.434 305
Figure 7a-4: Example data for Collection Info
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Data Submi ssi on - Types
There are two types of submissions: New Submission and Update.
A new submission is what is done every time new records are added to a project. Update submissions are for modifying records that
already exist in a project. If you wish to only update one or two records, please manually select the specimen from the species record
listing in your project and clicking on the edit button in the upper right corner. Any details can be edited in this way, except for adding
new taxonomy to BOLD. If there is new taxonomy to add to the BOLD library this should be sent in as an update.
Update Submission
The quickest way to update data is to download the Data Spread-
sheet from BOLD containing the records that need to be modi-
ed. To do so, click on Data Spreadsheets from the Downloads
menu on the upper left side of your project. Only download the
worksheets and records that will be affected by the update (e.g. if
the taxonomy needs to be updated only download the Taxonomy
worksheet, if specimen details and collection date need to be
update only download the Specimen Details and Collection Data
worksheets, etc.). Once the worksheets are downloaded, modify
the data and copy it into the standard submission spreadsheet.
The submitted update should reect what the data should be on
BOLD. Please send this on to the data manager.
NOTE: Any elds left empty will be considered blank and thus
removed from BOLD during an update. Do not remove any
data from the update sheet if youd like it to stay on BOLD. The
computer cannot distinguish between blank: do not update this
eld or blank: delete the content of this eld.
Updates to Voucher Info are slightly different from updates to
Taxonomy, Specimen Details, and Collection Data.
a.) Updates to Voucher Info
Identical to new submissions, updates to the voucher info are
project specic. The records need to be split into their corre-
sponding project.
b.) Updates to Taxonomy, Specimen Details, and Collection Data
Updates to taxonomy, specimen details, and collection data are
project independent. Records from any number of projects can
be submitted in one submission spreadsheet, and the number of
records are (in theory) innite for this type of update.
Please see the previous page for an example of the lled in
spreadsheet.
New Submission
New submissions are project specic, so that their data can be
associated with a project on BOLD. If records are submitted that
need to be entered into different projects on BOLD, a separate
le for each project needs to be sent.
The minimal requirements for a new submission on BOLD are:
Voucher Info Page - Sample ID
Voucher Info Page - Field ID and/or Museum voucher ID
Voucher Info Page - Institution Storing
Taxonomy Page - Phylum
Other useful information:
It is important to use a unique and original format for the sample
IDs. If the sample IDs provided are not original to BOLD, they
will need to be changed before the data can go online.
Provide as much detail and additional information as possible
with a new submission. That way, it will take less time later to
update the blanks.
Only the following characters may be used in the sample ID, eld
ID, and museum ID: Numbers, letters, and ^ . : - _ ( ) #
All other characters will be removed.
If the specimen has sex, reproduction or life stage values that
do not t the accepted values for Specimen Details, then please
move the information to the Extra Info or Notes elds.
In the case where the donor or identier is deceased or retired,
please make note of that in the email eld. This is important to
provide this information so we can keep the database up-to-
date.
If the submission is part of a campaign, iBOL Working Group, or
a checklist, please let us know in the submission email.
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7b) I mage Submi ssi on Protocol
Image File * Complete (incl. extension) and identical le
name (case sensitive) of images.
Original
Specimen *
Enter yes if the image shows the actual speci-
men for this record. Otherwise enter no.
View
Metadata *
A short tag describing the orientation of the
image that will appear on BOLD.
Caption Additional information about the image.
Copyright info or descriptions are recom-
mended.
Measurement Measurement that was taken (including the
unit of measurement.
Measurement
Type
Item that was measured (e.g. body length,
wing span, etc.)
Sample ID * Sample ID for photographed specimen, must
match Sample.
Process ID * Process ID for photographed specimen, must
match Process ID in BOLD.
Figure 7b-1: Image Submission Spreadsheet (ImageData.xls) completed with sample data.
This protocol outlines the image submission process on BOLD.
It describes the necessary format of the images and the ancillary
data, and the steps required to build the uploadable package re-
quired for a successful submission.
The image submission package for BOLD is a zip le containing a
set of images and an Excel spreadsheet that associates the neces-
sary data with each image. There must be a row in the spread-
sheet for each image uploaded and the required columns must
be lled in (See Table 1). A template spreadsheet can be down-
loaded from the BOLD site (www.boldsystems.org/dsfsdfsd)
The recommended steps are oulined below:
1. Collect Images:
Collect high-quality images of specimens in .jpg format for your
project. BOLD accepts high resolution images up (up to 20
megapixels) but only displays a greatly reduced thumnail. Your
high resolution image is archived but will not be used without
the submitters consent. Refer to the following page for a guide
on picture orientation and quality.
2. Assemble Package:
The image submission package should consist of all images (.jpg)
and a spreadsheet with the le names and ancillary data. Make
sure that all images in the package are accounted for in the
spreadsheet. When submitting more than one image per speci-
men simply copy the Sample ID and Process ID to the next line
with the le name of the consecutive image. You can upload 1 to
10 images per specimen, depending on organism characteristics.
Please photograph several different orientations if needed.
The submission spreadsheet should be named ImageData.xls and
contains the columns described in Table 1.
Image File
Original
Specimen
View
Metadata
Caption Measurement
Measurement
Type
Sample Id Process Id
ROM101912-D.JPG yes Dorsal skull 15 mm skull length ROM 10912 BM272-03
ROM101912-L.JPG yes Lateral lower jaw 7 mm length ROM 10912 BM272-03
ROM101912-L2.JPG yes Lateral skull 15 mm skull length ROM 10912 BM272-03
ROM101912-V.JPG yes Ventral skull 15 mm skull length ROM 10912 BM272-03
ROM101912-D2.JPG yes Dorsal skin 50 mm dody length ROM 10912 BM272-03
ROM101912-V2.JPG yes Ventral skin 50 mm body length ROM 10912 BM272-03
ROM101944-D.JPG no Dorsal skull 17 mm skull length ROM 10944 BM278-03
Steps:
A. Fill in the ImageData.xls data sheet with all the data related
to the images in the submission package.
To create the list of image les in a folder, open a terminal win-
dow (Start > Run > cmd in Windows), navigate to the folder
containing the image les, and then run one of the following
commands:
Windows dir /b *.jpg>list.txt
MacOS ls *.jpg*.JPG>list.txt
Linux/Unix ls *.jpg*.JPG>list.txt
These commands will generate a list of all the les in the cur-
rent folder and save it in list.txt. You can then open list.txt in
move the data into the Image File column.
* Required Fields
Table 7b-1: Field denitions for accompanying
spreadsheet.
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I mage Submi ssi on - Ti ps and Troubl eshooti ng
Zipped le must be under 195MB in size. If the upload fails to initialize, the zipped le
may be too large. Break it into two uploads, each with its own spreadsheet.
The spreadsheet can not contain any formulas.
If the upload program can not nd the image les, it is possibly because it can not read
the names. Make sure that the spreadsheet contains text values only.
Full lenames must be used in excel sheet. The extension (.jpg) must be included in the
image le name. The le extension is case sensitive.
Spreadsheet must be named ImageData.xls. If the upload program can not nd the excel
sheet, conrm that it is named correctly (case sensitive).
Max of 30 characters in the free text elds of the excel sheet. Verify that the data length
in these elds and make adjustments if necessary
Data must start on the second line of the spreadsheet. There is only one line for the
column headers.
Adding extra columns to the sheet will cause errors.
This section describes the most commonly-encountered image upload problems.
B. These two components (Image les and Spreadsheet) need to be placed in a single folder. Compress them all into a single le
before submitting. The following free tools are available to provide this functionality:
WinZip - http://www.winzip.com
WinRar - http://www.rarsoft.com
MacZipIt - http://www.maczipit.com
C. BOLD will accept a maximum le size of 195 MB. Upload the images to BOLD by clicking on the link Specimen Images in the
Uploads menu of the desired project. Select the zipped folder of images and then hit submit.
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Photography Gui de
Please take pictures using the high quality mode on your
camera. The specimen should be centered in the image
frame. Photos should be taken as close-up as possible, leaving
very little gap around the edges. The following standard
orientations should be adhered to when appropriate.
All images should be in landscape orientation, with a 2x3
aspect ratio. If your specimens do not easily t these
criteria please try to keep them in a standardized position.,
as this makes it much easier to compare specimens within
a project. If desired, a measurement scale may be included
in the image to provide a size reference.
Dorsal
The anterior of the specimen should be facing the top of the
image frame
The specimen should be face-down, with the dorsal aspect of
the head visible
Lateral
The anterior of the specimen should be facing the left side of
the image frame
The specimen should be oriented with the feet towards the
bottom of the image
Ventral
The anterior of the specimen should be facing the top of the
image frame
The specimen should be face-up, with the ventral aspect of the
head visible
Lateral
Dorsal
Ventral
Figure 7b-2: Suggested sample photographs.
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Table 7c-1: Field denitions for accompanying
spreadsheet.
Trace File * Complete (incl. extension) and identical
le name (case sensitive).
Score File Complete (incl. extension) and identical
le name (case sensitive).
PCR Primers
Fwd/Rev *
Primer codes are case sensitive.
Sequence Primer Primer codes are case sensitive.
Read Direction * Forward or Reverse.
Process ID * Process Id of specimen, must match
Process Id in BOLD.
7c) Trace Fi l e Submi ssi on Protocol
This protocol assists in the submission of trace les to BOLD. It
describes the necessary format of the les and the ancillary data
that is required for the correct submission.
1. Register Primers:
Please see the next page for details on how to register primers.
The submission package consists of trace les (.ab1), correspond-
ing phred les (.phd.1) and a spreadsheet with the le names
and ancillary data. The submission spreadsheet should be named
data.xls and contain the columns described to the right.
Trace File Score File
PCR
Fwd
PCR
Rev
Seq Primer Read Direction Process Id
KKBNA001-04_H01.ab1 KKBNA001-04_H01.phd.1 BirdF1 BirdR1 BirdR1 Forward KKBNA001-04
KKBNA001-04r_H07.ab1 KKBNA001-04r_H07.phd.1 BirdF1 BirdR1 BirdR1 Reverse KKBNA001-04
KKBNA002-04_G01.ab1 KKBNA002-04_G01.phd.1 BirdF1 BirdR1 BirdR1 Forward KKBNA002-04
KKBNA002-04r_G07.ab1 KKBNA002-04r_G07.phd.1 BirdF1 BirdR1 BirdR1 Reverse KKBNA002-04
KKBNA003-04_F01.ab1 KKBNA003-04_F01.phd.1 BirdF1 BirdR1 BirdR1 Forward KKBNA003-04
KKBNA003-04r_F07.ab1 KKBNA003-04r_F07.phd.1 BirdF1 BirdR1 BirdR1 Reverse KKBNA003-04
KKBNA004-04_E01.ab1 KKBNA004-04_E01.phd.1 BirdF1 BirdR1 BirdR1 Forward KKBNA004-04
Figure 7c-1: Trace File Submission Spreadsheet (data.xls) completed with sample data.
Steps:
A. Fill in the data.xls sheet with all the data about your les.
To create the list of the les in a folder, you need to open a ter-
minal window (Start > Run > cmd in Windows), navigate to the
folder where the trace and score les have been placed and then
run one set of the following commands:
Windows dir /b *.ab1>ab1.txt and dir /b *.phd.1 >phd.txt
MacOS ls *.ab1>ab1.txt and ls *.phd.1 > phd.txt
Linux/Unix ls *.ab1>ab1.txt and ls *.phd.1 > phd.txt
These commands will generate lists of all the les in the current
folder and save it ab1.txt and phd.txt. You can then open the text
les and move the data into the appropriate columns.
B. These components (Trace les, Score les and Spread-
sheet) need to by placed in a single folder. Compress them all
into a single le before submitting. The following free tools are
available to provide this functionality:
WinZip - http://www.winzip.com
WinRar - http://www.rarsoft.com
MacZipIt - http://www.maczipit.com
C. BOLD will accept a maximum le size of 195MB. Upload
the images to BOLD by clicking on the link Trace Files in the
Uploads panel of the desired project. Select the zipped folder
of les and then hit submit.
* Required Fields
BOLDSYSTEMS.org
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Trace Fi l e - Pri mer Regi strati on
Be sure that your primer codes are
registered with BOLD before assem-
bling the submission package. To regis-
ter your primers, select Register Prim-
ers from the Project Options menu in
your project on BOLD.
On the form, you are asked to ll in
the following information:
Figure 7c-2: BOLD Primer submission form
Primer Code Create a code for your primer. If the
primer is already published in a manuscript,
please use the code that is in press.
Primer
Description
This eld is for lling in a description of
what the primer is used for.
Alias Codes Fill in any other known code names for
your primer, separated by commas
Target Marker Select the target marker from the
controlled list of markers (e.g. ITS, COI
5, matK, etc.)
Primer Sequence Fill in the sequence, 5 to 3
Direction Select the direction
Reference/
Citation
Fill in references and/or citations
Notes Notes about the primer
Publicly Available If the primer has already been published,
or if you wish to make it publicly available,
this should be left public
If the primer you used has already been
registered under a different name, you
will be provided with the registered code
to be used in your submission.
Table 7c-2: Field denitions for accompanying gure.
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Trace Fi l e Submi ssi on - Ti ps and Troubl eshooti ng
Primers must be registered before upload. If the
primers are not registered, there will be an error.
Please refer to the previous page for details on
how to register primers.
Zipped le must be under 195MB in size. If the
upload fails to initialize, it is probably because the
zipped le is too large. Try breaking it into two
uploads, each with its own spreadsheet.
The spreadsheet cannot contain any formulas.
If the upload program can not nd the les, it
is possibly because it can not read the names.
Make sure that you have text values only in the
spreadsheet.
Full lenames must be used in excel sheet. The
extension (.ab1, .phd.1) must be included in the le
name. These extensions are case sensitive.
Spreadsheet must be named data.xls. If the upload
program can not nd the excel sheet, conrm that
it is named correctly (case sensitive).
Data must start on the second line of the
spreadsheet. There is only one line for the column
headers.
Do not add extra columns to the spreadsheet.
Trace les will not be downloadable from BOLD
until 24 hours after they have been submitted.
This section describes the most commonly encountered trace le upload problems.
Figure 7c-3: A list of public primers available from the project
console. These are helpful for those who are new to barcoding.
Figure 7c-4: Trace le for Vulpes vulpes (red fox).
BOLDSYSTEMS.org
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The sequence submission should consist of
sequences in fasta format referenced by BOLD
Process IDs.
2. Upload Package:
You can put up to 1000 sequences into one up-
load. Upload the sequences to BOLD by clicking
on the link Sequences in the Uploads menu of
the desired project. Paste the sequences into the
text box and hit submit.
If you wish to replace a sequence on BOLD, simply upload the new one with the same Process ID.
If you wish to delete a sequence on BOLD, simply upload NNNNN associated with the process ID.
7d) Sequence Submi ssi on Protocol
This protocol outlines the sequence le submission process on BOLD. It describes the necessary format of the
sequences, and the steps required for a successful submission.
Example:
>TZBNA001-05
CTGCAGGANCAAAAAATGAAGTATTTAAATTTCGATCTGTTAATAATATAGTAATAGCTCCTGCTAATA-
CAGGTAAAGATAATAATAATAAAAAAGCTGTAATTCCTACAGCTCAAACGAAAAGGGGTAGTTGATC-
GAAAAATATATTATTTAATCGTATATTAATAATAGTTGTAATAAAATTAATTGCTCCTAAAATAGAAGAA
>TZBNA002-05
CAGCTAATACGGGTAAAGATAATAATAATAAAAAAGCTGTAATTCCTACTGCCCAAACAAAAA-
GAGGTAATTGATCAAAAAATATATTATTTAAGCGTATATTAATAATAGTTGTAATAAAATTAATTGC-
CCCTAAAATAGAAGAAATTCCTGCTAAATGAAGAGAAAAAATAGCTAAATCTACAGAACTACCCCCAT-
GGGCGATATTAGAAGATAATGGGGGGTAGACTGTTCATCCTGTT
>TZBNA012-05
AAAATAGCTAAATCAACTGAGCTTCCTCCATGAGCAATATTAGATGATAGTGGGGGGTAAACTGT-
TCATCCTGTTCCAGCTCCATTTTCTACCACTCTTCTTGAAATTAAAAGAGTAATAGAAGGGGGGAG-
TAATCAAAATCTTATATTATTTATTCGTGGGAAAGCN
Figure 7d-2: Illustrative barcode for Homo sapiens (human).
Figure 7d-1: Pop-up window for uploading traces
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Once your project has been populated with the data,
images, traces and sequences that you have uploaded to
BOLD, it will look like the gures on the right. For further
information on how to navigate a project, please refer to
the description below.
Project Console
The console shows you a report of the amount of specimens,
along with tallies of any missing components of the records. The
console includes graphs to provide a quick visual overview of
the project, as well as a list of all the users on the project. The
links to the left provide access to uploads, downloads and various
analysis tools. The record listing can be accessed by clicking on
View All Records under the Project Data Views menu in the
upper left corner.
Record List
The record list gives access to the individual specimen and
sequence data for each record. You can select specic records
for analysis or updates using the checkboxes. Icons will appear
next to a record to indicate the presence of certain aspects of
a record.
Table 8-1: BOLD Record List icons
GPS coordinates present for sample
Images present for sample
The number of traces present
Stop codons present in sequence
Contamination present in sequence
Flagged record, not in ID engine
Click on the Sample ID or the Process ID to access the Specimen
Data and Sequence Data respectively, for each record
Specimen Window
This window provides voucher details, taxonomy, specimen
details and collection data, along with a world map of where
the specimen was collected. The images for the specimen are
located at the bottom of the window. To edit any details, simply
select Edit from the upper right corner.
Sequence Window
The sequence page gives access to various details about the trace
les and sequences for the specimen. Trace les can be viewed
or downloaded from this window. If desired, the ID engine can
be used to identify the sequence.
Near the bottom of the page is an illustrative barcode of
the species, along with a link to the Laboratory Information
Management System (LIMS) for the Canadian Centre for DNA
Barcoding.
Figure 8-2: BOLD Record List
Figure 8-3: Specimen Data Figure 8-4: Sequence Data
Figure 8-1: BOLD Project Console
8. BOLD Consol e
BOLDSYSTEMS.org
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Notes
Bi odi versi t y I nsti tute of Ontari o
Uni versi t y of Guel ph
579 Gordon Street
Guel ph, Ontari o, Canada
N1G 2W1
Copyr i ght 2008 Bi odi ver si t y I nst i t ute of Ont ar i o
Last modi f i ed: Oct 2008
ersi t y I nsti tute of Ontari o
BOLDSYSTEMS.org

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