Ion AmpliSeq
™
HD library chemistry:
• To calculate the Minimum Read
Count Per Sample parameter that
you enter during assay setup,
estimate the coverage depth that you
require to achieve the limit of
detection (LOD) needed for your
samples. The limit of detection
depends on coverage depth and
amount of input material.
For more information, see the Ion AmpliSeq
™
HD Library Kit User Guide (Pub. No.
MAN0017392).
Examples:
– Your panel has 200 amplicons in each of two primer pools, and you require
5% LOD and 1,400 reads/amplicon for a solid tumor sample.
Minimum Read Counts Per Sample =
(200 amplicons × 2 pools) × 1,400 reads/amplicon/sample = 560,000
– Your panel has 100 amplicons in each of two primer pools, and you require
0.1% LOD and 50,000 reads/amplicon for a cfDNA sample.
Minimum Read Counts Per Sample =
(100 amplicons × 2 pools) × 50,000 reads/amplicon/sample = 10,000,000
Note:
These calculations are based on 100% uniformity and 100% on target reads.
Depending on the actual uniformity of the panel, the quality of input sample, and
panel on-target percentage, minimum read counts per sample may need adjustment.
• Refer to the information in the following table for entering the number of library
amplification cycles for a custom Ion AmpliSeq
™
HD panel.
Primer pairs per pool
Recommended number of cycles
12–500
20
501–1,000
19
1,001–2,000
18
2,001–5,000
17
LOD
Minimum amplicon
coverage
5%
1,400
1%
7,000
0.5%
14,000
0.1%
50,000
Appendix C
Supplemental information
Guidelines for using custom assays with the Genexus
™
Integrated Sequencer
C
Genexus
™
Integrated Sequencer User Guide
137
