Access Array System for the Ion Torrent PGM Sequencing System: User Guide
24
Chapter 5: Bidirectional Amplicon
Tagging on the LP 48.48 IFC
This protocol outlines the bidirectional amplicon tagging strategy for the Ion
Torrent™ PGM™
Sequencer - 96
for amplicon libraries that have been generated on the Access Array™ system.
The goal of this protocol is to sequence both ends of an amplicon with a single-read
sequencing run. In the bidirectional amplicon tagging approach, tagged target-specific
primer pairs are combined with two sets of sample-specific barcode primer pairs. The
sample-specific barcode primer pairs are comprised of common sequence tags CS1 or CS2,
appended with the Ion Torrent adapter sequences (A and P1,
Table 6
). This approach
requires only one set of target-specific primer pairs, while the sample-specific barcode
primers are universal and can be used in multiple experiments.
In the figure that follows, you can see that bidirectional amplicon tagging generates two
types of PCR products per target region: one PCR product that allows for sequencing of the
5
' end of the target region (Set A) and one PCR product that allows for sequencing of the
3
'
end of the target region (Set B). Because both PCR products are clonally amplified onto the
Ion Sphere™ Particle (ISP) at the same time, one single-read sequencing run will yield
sequence information for both ends of the target region. Bidirectional sequencing will
produce high quality reads from both ends and across the full length of the amplicons.
Table 6. PCR products generated from b
idirectional amplicon tagging
This strategy uses a two-step PCR approach: (1) The first PCR is run on the IFC in the
presence of tagged, target-specific primers. (2) The harvested PCR product pools are then
split into two subsequent PCR reactions with two sets of sample-specific barcode primers,
Set A and Set B. Each is run on an independent 96-well plate. In Set A, the PCR reaction
products that are generated allow for sequencing of the 5' end of the target region and
utilize the A_BC_CS1 and P1_CS2 barcode primer combination. In Set B, the PCR reaction
products that are generated allow for sequencing of the 3' end of the target region and
utilize the A_BC_CS2 and P1_CS1 barcode primer combination.
Primer
Sequence
A_BC_CS1
5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-[BC]-ACACTGACGACATGGTTCTACA-3'
P1_CS2
5'-CCTCTCTATGGGCAGTCGGTGATTACGGTAGCAGAGACTTGGTCT-3'
A_BC_CS2
5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-[BC]-TACGGTAGCAGAGACTTGGTCT-3'
P1_CS1
5'-CCTCTCTATGGGCAGTCGGTGATACACTGACGACATGGTTCTACA-3'
