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Trace Submission Protocol

Trace files are necessary for records to qualify for Barcode status and also to provide quality control for sequences. As well, BOLD can store failed or low quality traces that did not produce a quality sequence as part of an audit trail.

This protocol assists in the submission of trace files consisting of electropherograms and optional Phred/score files to BOLD. It describes the necessary format of the files and the supporting data that is required for the correct submission. Before traces can be uploaded, specimen data records must first be uploaded to a BOLD project.

Uploading Trace Files
  1. Group Trace Files
    • Group trace files into a folder. Confirm that primers exist on BOLD by clicking on Primers under the Databases section of the BOLD header once signed in. If a primer isn't found, please view the Primer Registration Protocol for details on how to register a new primer in BOLD. Primer codes must match what is on BOLD exactly.
  2. Assemble the Excel template for Trace Submission
    • excel Trace File Submission Template (data.xls)
      This spreadsheet associates the trace files with the records on BOLD as well as uploads ancillary data such as primers, direction, and marker. All the traces must be accounted for in the spreadsheet to be uploaded. Additional traces that are not referenced in the Excel file will be ignored. BOLD accepts a maximum of 10 traces per record.
    • The submission spreadsheet must be named data.xls and contains the columns described in the table below.
    • The minimal requirements on the Excel template are:
      • Trace File name
      • Forward and Reverse PCR Primers
      • Read Direction
      • Process ID
      • For a multimarker project, the marker must be filled in, otherwise traces will upload to the primary marker by default.
  3. Zip Package of Traces and Excel File
    • The two components, consisting of trace files and the spreadsheet need to be placed in a single folder. Compress them all into a single zipped file before submission. The free tools listed below are available to provide this functionality, however, most modern operating systems have built-in functionality for zipping:
      • WinZip: http://www.winzip.com
      • WinRar: http://www.rarsoft.com
      • MacZipIt: http://www.maczipit.com
    • BOLD will accept a maximum zipped file size of 190 MB. Please confirm the file size before moving to Step IV. If the file is too large, break it into more than one upload, each with its own spreadsheet.
  4. Submit file to Project
    1. Login to BOLD and click on Trace Files under the Data Uploads menu in the User Console. This option is available to project managers and project users with editing sequence access.
    2. In the form, select the Zip file to submit, and the run site for the analysis. Users can also include brief description if desired.
    3. Click on Submit. Do not close the pop-up window until the successful upload message is displayed. If phred scores are not included in the submission, BOLD will automatically calculate the quality values for the traces. Traces will not be viewable or downloadable from BOLD until the next business day.

excel Trace File Submission Template (data.xls)

Field definitions for accompanying spreadsheet. *Required Fields
Trace File* Complete name, including extension (.ab1 or .scf) and identical to file name in the folder.
Score File Complete name, including extension (.phd.1) and identical to file name in the folder, if available.
PCR Primers Fwd/Rev* Primer codes are case sensitive. Both must be filled in.
Sequence Primer Primer used in the sequencing process.
Read Direction* Forward or Reverse.
Process ID* Process ID of record, must match Process ID on BOLD.
(2 columns must be left blank
after the Process ID column)
The marker needs to be filled in with the appropriate code:
  • COI-5P
  • ITS
  • rbcL
  • matK
  • etc...
Example Submission Spreadsheet for Trace File Upload:
Trace File Submission Spreadsheet (data.xls) completed with sample data.
Trace File Score file PCR Fwd PCR Rev Seq Primer Read Direction Process ID (blank) (blank) Marker
KKBNA001-04_H01.ab1 KKBNA001-04_H01.phd.1 BirdF1 BirdR1 BirdF1 Forward KKBNA001-04 COI-5P
KKBNA001-04r_H07.ab1 KKBNA001-04r_H07.phd.1 BirdF1 BirdR1 BirdR1 Reverse KKBNA001-04 COI-5P
KKBNA002-04_G01.ab1 BirdF1 BirdR1 Forward KKBNA002-04 ITS
KKBNA002-04r_G07.ab1 BirdF1 BirdR1 Reverse KKBNA002-04 ITS
Trace File Submission - Tips and Troubleshooting
  • Primers must be registered before upload. If the primers are not registered, there will be an error. Please view the Primer Registration Protocol for more details.
  • The zipped file must be under 190MB in size. If the upload fails to initialize, it is probably because the zipped file is too large. Try breaking it into more than one upload, each with its own spreadsheet.
  • The spreadsheet cannot contain any formulas. If the upload program cannot find the files, it may be because it cannot read the names. Make sure to have text values only in the spreadsheet.
  • Full filenames must be used in the excel template. The extension (.ab1, .scf, .phd.1) must be included in the file name. These extensions are case sensitive.
  • The spreadsheet must be named data.xls. If the upload program can not find the excel sheet, confirm 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.
  • If trace files appear under the wrong marker, or the marker name was entered improperly, then the program will not be able to read the marker column and will place the trace files by default under the primary marker in the project. To remove primers from the system, please forward the excel sheet to support@boldsystems.org.
  • Trace files will not be downloadable or viewable from BOLD until 24 hours after they have been submitted.
  • Never rename .scf files as .ab1 files as this conversion will lose data from the trace files. BOLD supports both file types.
  • Phred files (.phd.1) are not required for a trace upload. If Phred files are not uploaded, BOLD will calculate the Phred scores automatically.
To obtain Trace File names from a folder:

To create the list of trace files in a folder, open a terminal window (Start > Run > cmd in Windows), navigate to the folder containing the trace and score files, and then run one of the commands listed below:

  • Windows dir /b *.ab1>ab1.txt or dir /b *.scf>scf.txt and dir /b *.phd.1 >phd.txt
  • MacOS/Linux/Unix ls *.ab1>ab1.txt or ls *.scf>scf.txt and ls *.phd.1 > phd.txt

These commands will generate a list of all the files in the current folder and save it in a document called ‘ab1.txt’ or 'phd.txt' that will appear in the current folder. Open this text file and copy the data into the appropriate columns of the Excel spreadsheet. If the file endings do not appear, uncheck the "Hide Extensions for Known File Types" option in your computer.

To obtain Process IDs from BOLD:

Obtain the Process IDs by clicking on Data Spreadsheets under the Downloads menu on the left side of a Project Console. Download the Progress Report to get the Process IDs that are assigned to each Sample ID.

Trace Updates or Deletions
  • Users can upload more traces in separate batches to any record at any time.
  • To update details on a trace file, if for example the wrong primers were uploaded, users can just re-upload the same trace file to the system with the corrected information in the spreadsheet.
  • To delete any traces for a record, contact the BOLD Support Team through support@boldsystems.org

For any questions or concerns about trace submissions or management on BOLD, please email the Support Team through support@boldsystems.org

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  • tag_submission
  • tag_tracefile
  • tag_project

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