The Illumina NextSeq 500/550 High Output Kit (1× 75 cycles; Illumina catalogue nr. 20024906) can be used for digitalMLPA™ on an Illumina NextSeq 500 or 550 sequencing system. However, since digitalMLPA requires single-end reads of 110 nt, sequencing needs to be performed with two custom sequencing primers.

You can order the custom sequencing primers from a supplier of choice (at 100 nmol scale, desalted). They cannot be ordered from MRC Holland.

Read 1 primer MRC-NS-R1:      5'-TGTGCCAGCACGATCCAATCTCGCATA-3'
Index read primer MRC-NS-IR:  5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'

 

Instructions

Sequencing primer preparation

• Make stock primer solutions by dissolving the custom sequencing primers in TE buffer to a concentration of 100 µM. Aliquot and store at -20 °C.
• Before use, thaw the two stock primer solutions and mix by vortexing briefly. Prepare a 0.3 µM dilution of each sequencing primer:
  • Mix 6 µl 100 µM MRC-NS-R1 + 2 ml HT1 Hybridization Buffer in a tube labelled "well 7".
  • Mix 6 µl 100 µM MRC-NS-IR + 2 ml HT1 Hybridization Buffer in a tube labelled "well 9".
• Thaw a sequencing cartridge and wipe the foil seal of wells 7 and 9 with a low-lint tissue. Pierce the foil seal of wells 7 and 9 with a clean pipette tip and add 2 ml of diluted sequencing primers to the respective wells.

NextSeq run instructions

Standalone mode

• In the Run setup screen of the Illumina NextSeq Control Software (NCS), click Create Run and select Generate FASTQ.
• Specify custom primers on the run setup screen: Custom read 1 primer; Custom index 1 primer.
• Use the following run settings:
  • Library prep kit: TruSeqLT.
  • Read type: single read.
  • Read length: 75 nt.
  • Index read length: 10 nt.
• Under Sample ID, enter a name (can be the experiment name) and add as sequence: NNNNNNNNNN (N10).
• Click Finish.

Basespace or Basespace Onsite

• Specify custom primers on the Basespace Prep tab.
• Select the checkbox for R1 and the index read (custom primer for read 1 and custom primer for the index read).

Demultiplexing the digitalMLPA reads

• Demultiplexing is done using Illumina's Linux-based bcl2fastq Conversion Software. The software converts raw Illumina BCL files into FASTQ files along with performing data demultiplexing.
• Download the software.
• Additional information about the software can be found on this website.
• If needed, contact us for barcode sequence files.

Coffalyser digitalMLPA

• Select a barcode lot that includes the phrase Illumina demultiplexed, such as 03-xxx-yymmdd ; Illumina demultiplexed FastQ (e.g. NextSeq 75 nt reads).
• Follow all other steps as described in the Coffalyser digitalMLPA User Manual.