BaseSpace Sequence Hub Prep Tab How to create a custom library prep kit

The steps outlined in this bulletin are intended for customers generating sequencing data on a NextSeq500/550 or MiniSeq instrument, and using BaseSpace Sequence Hub for data analysis, which requires the use of the Prep Tab for setup.

• Note: These directions are not applicable for use with Run Planning in BaseSpace.

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 Important considerations 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.

1. [Index1Sequences]: In the [Index1Sequences] section, list the names of the indexes in the first column, and the associated sequences in the second column.

2. [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.

3. [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.

4. [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—enter up to 12 values in this section. Adding more than 12 column values will disrupt the well display in the Library setup.

1. [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.

• Note: Adding more than 8 row values will disrupt the well display in the Library setup.

1. Save the changes to the file.

2. Log in to BaseSpace Sequence Hub, select your name at the top right and select Settings.

3. In the Settings section select Library Prep Kits.

4. Select Prep Tab from the drop-down.

5. Select New Library Prep Kit.

6. 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, select Choose .csv File.

7. Select the CSV file saved in step 9, then select Create New Kit.

To delete a custom kit for Prep Tab, open your account dropdown and select Resources. Select the Prep Tab Kits option, and then select the X next to the kit you want to delete. Default Illumina kits cannot be deleted.

Important considerations:

• All values in the [DefaultLayout_ByWell] and [DefaultLayout_ByColumn] sections must be filled in. 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.

• Some index names, especially ones containing well positions, can result in the plate graphic failing to render. If this happens, use more generic names as provided in the kit template.

• Dual-index libraries support up to 96 unique index combinations using a maximum of 12 first indexes and 8 second indexes. Dual-index kits using unique combinatorial indexes are not supported as custom kits.

• 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.

For any feedback or questions regarding this article (Illumina Knowledge Article #1770), contact Illumina Technical Support [e-mail].