Guidelines for Running Samples on the Illumina MiSeq (E7000)

The NEBNext Direct protocol incorporates Illumina adapter sequences; therefore, the libraries generated from this protocol may be sequenced on Illumina platforms including the MiSeq, NextSeq, and HiSeq Platforms. Here we describe the steps necessary to sequence NEBNext direct libraries on the Illumina MiSeq.

If samples are run on the Illumina NextSeq, please note that while the i5 index is generated in the reverse complement orientation, no adjust-ments need to be made because the i5 UMI is a random sequence.

4.1. Follow the Illumina Miseq Reporter Software Guide to reconfigure the MiSeq reporter parameter CreateFastqForIndexReads to write both index reads to FASTQs.

The sample barcode is in the i7 position and is sequenced as the index read 1. The UMI is in the i5 position and is sequenced as the index read 2. By default, the Illumina MiSeq is set to not generate FASTQs for index reads; therefore, it is necessary to override this setting in order to make use of these features.

4.2. Generate a MiSeq sample sheet with the following parameters:

Workflow: Generate FASTQ
Application: FASTQ only
Assay: TruSeq HT
Chemistry: Amplicon

The remaining fields will require information specific to your experi-ment. The choice for read lengths will depend on the particular bait design for your experiment, but in general NEBNext Direct baits are designed for PE75 reads. PE150 reads can also be used to increase depth of coverage. For a sample sheet template, visit the NEBNext Direct website at: - NEBNext Direct Cancer HotSpot Panel (NEB #E7000)

4.3. Pool, dilute and denature samples for an 8 pM final concentration following the MiSeq Denature and Dilute Libraries Guide and the NEB-Next Direct guidelines for pooling together multiple barcoded samples (

The number of samples that can be pooled together will depend on the input amount, panel size, and number of reads required from each sample for the particular analysis being performed.

4.4 Follow the MiSeq System Guide to load your samples and run the MiSeq

4.5 For information on bioinformatic utilization of unique molecule indexes (UMI's) please refer to Using Unique Molecular IDs with NEBNext Direct – Data Usage Guideline Page within the "Other Tools & Resources" tab at: