Available Illumina enrichment analysis workflows, including those in
Run Manager, and MiSeq
Reporter, can use either the Illumina fixed panel manifests to
specify the targeted regions for variant calling or use a custom
manifest file. Manifests are provided for Illumina custom panels
created through Illumina DesignStudio.
Alternatively, many non-Illumina vendors provide BED
files to define the targeted regions that need to be converted into a
manifest. As shown in the image below, while the contents of a BED
file (top) and manifest (bottom) are similar, the manifest file has a
different format and some additional information. These examples are
taken from the Nextera
Rapid Capture Expanded Exome content set.
To convert a BED file into an enrichment manifest file, perform the
- Download the Nextera
Rapid Capture Expanded Exome manifest to use as a template.
Retain the [Header] section, including the path to the
- For the [Regions] section, copy
any target names from column 4 of the BED file to the Name column of
the manifest file (column 1). See 'Important Considerations' below
for more guidance.
- Copy the first, second, and third
columns from the BED file to the second, third, and fourth columns
of the manifest file (ie Chromosome, Start, End), respectively.
- If upstream and downstream probe lengths are provided by the
vendor, they can be included in the next 2 columns. If they are not
provided, set these values to 0.
- Save as a tab-delimited
text file (TSV), not CSV or Excel.
- Target names must be unique. To ensure that the names are
unique, they can be appended with a series of numerical values, such
as Target.001, Target.002, Target.003.
- Only letters,
numbers, periods, dashes, and underscores are allowed in the target
names. Spaces and special characters (such as, but not limited to :
and ") are not allowed. Remove any lines that contain track
information contained within < > symbols.
target names or the usage of unsupported characters return the error
message "[Probes] section is missing or invalid" when
importing into BaseSpace Sequence Hub. While enrichment manifest
files do not require a [Probes] section, this message indicates that
the file format is invalid.
- When importing a manifest file
to BaseSpace Sequence Hub, files larger than 30 MB in size may fail
the validation step.