Life Technologies
™
| Parkinson's cell models
14
Ion PGM
™
sequencing
To expedite detection and quantitation of TAL editing events,
we developed an amplicon sequencing method on the Ion
PGM
™
System (Figure 5). This method allowed us to generate
thousands of sequencing reads from any one sample and to
process up to 96 samples in one sequencing run.
With this method, we were able to determine whether an
isolated iPSC colony harbored any cells that contained a
desired mutation, whether that colony contained a clonal or
mixed population of cells, and whether a clonal population of
cells was homozygous or heterozygous for the mutation.
Figure 5. Workflow for Ion PGM
™
sequencing to detect and confirm editing events. The 400 bp amplicons used for the GeneArt
®
Genomic Cleavage Detection and TaqMan
®
SNP
Genotyping Assays served as the template for generating a 200 base pair amplicon for Ion PGM
™
sequencing. A library was prepared from the amplicon using the Ion Plus Fragment
Library Kit and Ion Xpress
™
Barcode Adapters. Template was then prepared from the library using the Ion OneTouch
™
2 System and Ion OneTouch
™
200 Template Kit v2 DL. The library was
then sequenced using the Ion PGM
™
200 Sequence Kit and the Ion PGM
™
System. The data were analyzed using software such as Integrative Genomics Viewer (IGV) [6].
Amplified region
of genome
Template preparation
Sequencing analysis
(e.g., IGV)
Ion Torrent
™
sequencing
using Ion PGM
™
System
Library preparation
