Guidelines for library quantification—library runs
• We recommend that you use libraries that are freshly quantified and diluted
before pooling in a library batch.
• Pre-prepared libraries can be quantified by one of the following three methods:
– Quantification using the Agilent
™
2100 Bioanalyzer
™
instrument
– Quantification using the Qubit
™
Fluorometer
– Quantification by qPCR using the Ion Library TaqMan
™
Quantitation Kit
See one of the following guides for specific procedures.
– Ion AmpliSeq
™
Library Kit 2.0 User Guide (Pub. No. MAN0006735)
– Ion AmpliSeq
™
Library Kit Plus User Guide (Pub. No. MAN0017003)
– Ion AmpliSeq
™
HD Library Kit User Guide (Pub. No. MAN0017392)
Dilute and pool libraries, and load the sample plate—library run
1.
Dilute each manually prepared and quantified sample library to 125 pM with
nuclease-free water.
Note:
Each library must be barcoded with a unique barcode or barcode pair. Use
this concentration as a starting point, then titrate up or down based on
sequencing results, if needed.
2.
Add equal volumes of each library to a new 1.5-mL low DNA retention tube so
that the total volume is greater than the volume specified in the run setup guide
provided by the run plan.
3.
Mix well by pipetting up and down five times, then transfer the specified volume
of each library batch to the sample plate position specified in the run setup
guide.
4.
Seal the plate with a sheet of Adhesive PCR Plate Foils (Thermo Fisher Scientific
Cat. No. AB0626).
Note:
The use of other plate seals may affect performance.
5.
Keep the plate on ice until you are ready to load it in the sequencer.
Chapter 6
Dilute the samples and load the sample plate
Guidelines for library quantification—library runs
6
Genexus
™
Integrated Sequencer User Guide
71
