meth_4.2.3 Illumina MiSeq 2 x 150 bp paired reads

Contributor(s): Ramaciotti Centre for Genomics, UNSW, Sydney

E.g. 18Sv9

1. Denature according to Illumina protocol, with increased PhiX control spike-in as recommended for low diversity libraries. See Preparing Libraries for Sequencing on the MiSeq (part #15039740).
2. Prepare MiSeq Reagent Cartridge (v2 300 – cycles). See MiSeq Reagent Kit v2 – Reagent Preparation Guide (part # 15034097).
3. Using an extra-long pipette tip set to 75 μL, add 3.4 µL of Read1 sequencing primer (100 μM) into well 12 of the MiSeq Reagent Cartridge and mix 10 times. Repeat adding the Index Primer into well 13 and the Read2 sequencing primer into well 14.
4. Load 600 μL of library pool into the MiSeq reagent cartridge in designated reservoir/sample well.
5. Modify sample sheet to include the appropriate primer’s sequence/indexes.

Start sequencing run following MiSeq System User Guide (part # 15027617).