meth_4.2.1 Illumina MiSeq 2 x 300 bp paired reads

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

E.g. B16S, A16S, ITS

1. Denature according to IIlumina protocol, with increased PhiX control spike as recommended for low diversity libraries. See Preparing Libraries for Sequencing on the MiSeq (part #15039740).
2. Prepare MiSeq Reagent Cartridge (v3 600 cycles). see MiSeq Reagent Kit v3 Reagent Preparation Guide (part # 15044983).
3. For B16S and ITS, add custom sequencing primers into reservoirs 12-14. See Using Custom Primers on the Miseq (part # 15041638). 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 appropriate primer’s sequence/indexes
6. Start sequencing run following MiSeq System User Guide (part # 15027617).