How do I create a custom library prep kit in BaseSpace Sequence Hub?

08/07/19


The steps outlined in this bulletin are intended for customers generating sequencing data on a NextSeq 500/550 or MiniSeq instrument, and using BaseSpace Sequence Hub for data analysis, which requires the use of the Prep tab for setup. The standard supported Illumina library prep kits are already included in the “Prep Libraries” section by default, but for non-Illumina libraries, a custom library prep kit must be created and uploaded through the Prep tab. Both dual- and single-index kits can be created. To add a custom library prep kit, perform the following steps:

  1. Download either the LibraryPrep_Template-DualIndex.csv or LibraryPrep_Template-SingleIndex-v2.csv file, as appropriate. There is not a template available for dual-indexed kits using unique combinatorial indexes (see note below).
  2. Open the file in either Excel or a plain text editor, such as Notepad.
  3. [Settings]: In the [Settings] section, list the adapter sequences to be used for adapter trimming. If no adapter trimming is desired, delete the AdapterSequenceRead1 and AdapterSequenceRead2 lines. Note: There is a 20-character limit for adapter sequences.
  4. [Index1Sequences]: In the [Index1Sequences] section, list the names of the indexes in the first column, and the associated sequences in the second column.
  5. [Index2Sequences]: For dual-index libraries only - In the [Index2Sequences] section, list the names of the second indexes in the first column and the associated sequences in the second column. Do not reverse complement the second index sequences—BaseSpace automatically reverse complements these sequences during demultiplexing.
  6. [DefaultLayout_ByWell]: Copy the index names from the first column in the [Index1Sequences] section and paste them into the second column of the [DefaultLayout_ByWell] section. Repeat this procedure until all the wells are listed.
  7. [DefaultLayout_ByColumn]: Copy the index names from the [Index1Sequences] section and paste them into the second column of the [DefaultLayout_ByColumn] section.
    • Note: For both single- and dual-index libraries only enter up to 12 values in this section. Adding more than 12 column values will disrupt the well display in the Library setup.
  8. [DefaultLayout_ByRow]: For dual-index libraries only - Copy the names from the first column of the [Index2Sequences] section and paste them into the second column of the [DefaultLayout_ByRow] section.
  9. Save the changes to the file.
  10. Log in to BaseSpace Sequence Hub and go to the account Settings section.
  11. Select Library Prep Kits from the Settings sidebar.
  12. Select New to upload the kit definition.
  13. In the screen that appears, name the custom kit and specify any other options, such as read types, indexing strategies, and default read cycles. For single-index libraries, clear the
  14. Choose the samples and click Prep Libraries.
  15. Select +Custom Library Prep Kit from the Library Prep Kit drop-down menu.
  16. In the screen that appears, name the custom kit and specify any other options, such as read types, indexing strategies, and default read cycles. For single-index libraries, clear the Dual Index checkbox. Then, click Choose .csv File.
  17. The new library prep kit is automatically selected, and appears in the drop-down menu for any future runs.

Important considerations:

  • All values in the [DefaultLayout_ByWell] and [DefaultLayout_ByColumn] sections, and for dual-index libraries only, the [DefaultLayout_ByRow] section, must be filled in. If using fewer than the available number of wells, columns, and rows, index names that have already been used can be repeated.
  • Dual-index libraries support up to 96 unique index combinations using a maximum of 12 first indexes and 8 second indexes.
  • Single-index libraries support up to 96 unique index sequences. An example of how to set it up can be found in this CSV file: LibraryPrep_Template-SingleIndex96-v2.csv
  • Only a single library type can be used at one time. All indexes must have the same length. Combining library types of different index lengths, multiple adapters, or single- and dual- indexes, is not supported.
  • All library prep kits can be viewed and deleted under the My Account section.