Guidelines for Running Samples on the Illumina MiSeq (NEB #E9500 and #E9530)

4.1. Guidelines for Running Samples on the Illumina MiSeq

The NEBNext Direct protocol incorporates Illumina adaptor sequences; therefore, the libraries generated from this protocol may be sequenced on Illumina platforms including the iSeq 100, MiSeq, NextSeq®, HiSeq® and Novaseq® platforms. Here we describe the steps necessary to sequence NEBNext Direct GS libraries on the Illumina MiSeq.

4.1.1. Prepare a MiSeq sample sheet: Download a MiSeq sample sheet, NEBNext Direct GS Barcodes file, and refer to the “Sample Sheet Guidelines” located in the “Protocols, Manuals & Usage” tab on the NEBNext Direct GS Target Enrichment product page at Do not use the Illumina Experiment Manager to generate a sample sheet. Fill in the sample sheet with your sample and barcode information. Transfer the sample sheet file (*.csv) to the MiSeq and save the file in D:\Illumina\Miseq Control Software\SampleSheets\

4.1.2. Pool, dilute, and denature samples for an 8-10 pM* final concentration following the MiSeq Denature and Dilute Libraries Guide and the Index Pooling Guidelines in Section 3 (page 17).

* Based on quantification with an Agilent High Sensitivity DNA Kit or Agilent High Sensitivity D1000 ScreenTape using the region between 100-1200 bp. Some optimization may be required for optimal cluster density.

4.1.3. Follow the MiSeq System Guide to load your samples and run the MiSeq. When prompted, upload the sample sheet that was prepared in 4.1.2.