Sample sheet creation for MiSeq Control Software v2.6 and Earlier

When creating a sample sheet for use with MiSeq Control Software (MCS) v2.6 and earlier:

• Illumina recommends creating the sample sheet using Illumina Experiment Manager before preparing sample libraries.

• Before starting the run, make sure the sample sheet is accessible to the instrument. Users may copy the sample sheet to a network location or copy the sample sheet from a USB drive using the "Manage Files" feature in MCS.

  • If copying from network or USB drive, it is best practice to store the file locally and reference the local file location during run setup.

• When the run begins, the software copies the sample sheet from the designated sample sheet folder to the root of the MiSeq Output folder. At the end of the run, the sample sheet is used for secondary analysis by the MiSeq Reporter software.

Sample Sheet Sections

• Naming the sample sheet: Name the sample sheet as the reagent cartridge barcode number associated with the sequencing run, and use a \.csv extension. The barcode number is located on the reagent cartridge label directly below the barcode. If the barcode number is not known, use a preferred name for the sample sheet followed by \.csv. In this case, the software will not automatically locate the sample sheet during run setup, and the user must manually indicated the sample sheet file path.

• Header Parameters: Investigator name, Project name, Experiment name, Date, Workflow (REQUIRED), Assay, Chemistry (REQUIRED).

  • For any workflows that require dual indexing, Chemistry field must be Amplicon.

  • For workflows that require single indexing, Chemistry field must be Default.

• Read Section parameters: Number of cycles Read 1 (REQUIRED), Number of cycles Read 2 (REQUIRED for Paired End runs). The index sequences defined in the Data section specify the number of cycles for the index reads.

• Column Headings: SampleID (REQUIRED), Sample_Name, Index, Index 2.

• Sample Sheet Settings: CustomRead1PrimerMix, CustomerRead2PrimerMix, CustomIndexPrimerMix, PercentTilesToScan (default value of 1=100% of tiles).

• Genome Folder Path: The GenomeFolder contains the reference genome in FASTA file format. For optimal results, store reference genomes on the local drive or use BaseSpace. Enter the full UNC path to the GenomeFolder in the sample sheet. Do not enter the path using a mapped drive (eg, X:). For instructions on locating the UNC Path on an instrument, refer to the Knowledge Base Article How to identify the UNC path of a network server or location.

• Adapter Settings: The Adapter sample sheet setting prevents reporting sequence beyond the sample DNA by trimming the specified sequence in FASTQ files. Trimming the adapter sequence avoids reporting of spurious mismatches with the reference sequence, and improves performance in accuracy and speed of alignment.

• Read Stitching: When set to true (1), paired-end reads that overlap are stitched to form a single read in the FASTQ file. At each overlap position, the consensus stitched read has the base call and quality score of the read with higher Q-score.

For more information about creating sample sheets, reference the MiSeq Sample Sheet Quick Reference Guide.

For MCS v3.1 and higher, sample sheet validation is done through Local Run Manager.

For any feedback or questions regarding this article (Illumina Knowledge Article #2396), contact Illumina Technical Support [e-mail].