241- Dilute library to ~100 pM, combine, then proceed to template
preparation, or libraries can be stored at 4–8 °C for up to
1 month. For longer term, store at − 20 °C. - The instrument Ion OneTouch 2 (Fig. 3a) should be installed
and set up properly. - Before each use, the amplification plate, reagent oil, recovery
solution, and plastic ware should be set up. - Each time a new kit was used, all plastic ware and reagents
should be changed to correspond with the kit currently in use. - Importantly the waste container was emptied before every run,
as any backwash would cause permanent contamination of the
entire instrument. - An amplification solution should be prepared and installed for
each sample pool (eight samples). - Before setting up the reaction, the Ion OneTouch 2× Reagent
Mix should be thawed and subsequently kept at 4 °C. - Also, the enzyme mix and preprepared library should be vor-
texed just before use. - In an Eppendorf tube, the following components should be
added in the designated order:
(a) 280 μL Nuclease-free water.
(b) 500 μL Ion OneTouch 2× reagent mix.
(c) 100 μL Ion OneTouch enzyme mix.
(d) 20 μL Diluted library pool.
3.2 Preparation
of Template
Fig. 3 Ion OneTouch 2 system. (a) Ion OneTouch 2 instrument. It helps in the automated delivery of the template
into ion sphere particles for sequencing. (b) Ion OneTouch Enrichment system. It helps in the enrichment of
recovered ion sphere particles for sequencing
Next Generation Sequencing and Esophageal Adenocarcinoma