The steps outlined in this bulletin are intended for customers
generating sequencing data on a NextSeq 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
- Download either the LibraryPrep_Template-DualIndex.csv
file, as appropriate. There is not a template available for
dual-indexed kits using unique combinatorial indexes (see note
- Open the file in either Excel or a plain text editor,
such as Notepad.
[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
Note: There is a
20-character limit for adapter sequences.
[Index1Sequences]: In the [Index1Sequences] section, list the
names of the indexes in the first column, and the associated
sequences in the second column.
[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.
[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.
[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.
[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.
- Save the changes to the file.
- Log in to BaseSpace
Sequence Hub and go to the account Settings section.
Library Prep Kits from the Settings sidebar.
- Select New to upload the kit definition.
- 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.
- Select the
CSV file saved in step 9, and then select Create New
- 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.
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.
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
supported as custom
- Single-index libraries support up to 96
unique index sequences. An example of how to set up the sample
sheet 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
- All library prep kits can be viewed, and custom kits deleted, in the
Library Prep Kits
section of the account settings