How to convert a custom BED file to a manifest file for enrichment analysis

Available Illumina enrichment analysis workflows, including those in BaseSpace Sequence Hub, Local 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, but 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.

Note: Manifests use 1-based genomic intervals while bed files uses 0-based genomic intervals.

To convert a BED file into an enrichment manifest file, perform the following steps:

• Download the TruSight Cancer Content Set Manifest File A to use as a template. Retain the [Header] section.

• For the [Regions] section, copy target names from column 4 of the BED file to the Names column of the manifest file (column 1). See the considerations below for more guidance.

• Add 1 to each value in the Start column (eg, 14362 becomes 14363). Copy the first, second, and third columns from the BED file (chromosome, target start, target end) to the second, third, and fourth columns of the manifest file (Chromosome, Amplicon Start, Amplicon 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.

• The Group column is informational can be left blank or deleted.

• Save as a tab-delimited text file (TSV), not CSV or Excel.

Important considerations

• Imported manifest files must be less than 30 MB in size to pass the validation step.

• 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.

Nonunique target names or the usage of unsupported characters return the error message "[Probes] section is missing or invalid.” While enrichment manifest files do not require a [Probes] section, this message indicates that the file format is invalid.

For any feedback or questions regarding this article (Illumina Knowledge Article #2840), contact Illumina Technical Support [e-mail].