Proceed to “Dilute the samples (if needed) and load the sample plate—sample
Guidelines for using custom assays with the Genexus
™
Integrated
Sequencer
Follow these guidelines for setting up assays on the Genexus
™
Integrated Sequencer if
you are using a custom Ion AmpliSeq
™
or Ion AmpliSeq
™
HD assay.
Ion AmpliSeq
™
library chemistry:
• For germline Ion AmpliSeq
™
assays, start with a coverage depth of 150 reads per
amplicon for calculating the Minimum Read Count Per Sample that you enter in
the Panel step of assay setup.
Example: Your panel has 500 amplicons in each of two primer pools.
Minimum Read Counts Per Sample =
(500 amplicons × 2 pools) × 150 reads/amplicon/sample = 150,000
• For somatic Ion AmpliSeq
™
assays, start with a coverage depth of 2,500 reads per
amplicon for calculating the Minimum Read Count Per Sample when you set up
your assay.
Example: Your panel has 500 amplicons in each of two primer pools.
Minimum Read Counts Per Sample =
(500 amplicons × 2 pools) × 2,500 reads/amplicon/sample = 2,500,000
• Refer to the information in the following table for entering the number of target
amplification cycles and anneal/extend time parameters for a custom Ion
AmpliSeq
™
panel.
Primer pairs per pool
Recommended number of
amplification cycles
(10 ng high-quality
DNA/RNA)
[1]
Anneal/Extend time
[2]
RNA fusion panels
28
4 minutes
12–24
22
4 minutes
25–48
21
4 minutes
49–96
20
4 minutes
97–192
19
4 minutes
193–384
18
4 minutes
385–768
17
4 minutes
769–1,536
16
8 minutes
1,537–3,072
15
8 minutes
3,073–6,144
14
16 minutes
6,145–24,576
13
16 minutes
[1]
Add 3 cycles for low quality (FFPE) samples.
[2]
For Ion AmpliSeq
™
panels using a 375-bp amplicon design, add 4 minutes to the anneal/extend time
recommended in the table.
Appendix C
Supplemental information
Guidelines for using custom assays with the Genexus
™
Integrated Sequencer
C
136
Genexus
™
Integrated Sequencer User Guide
