Before preparing libraries, record information about your samples using an option available for your sequencing system:
The AmpliSeq for Illumina Childhood Cancer Panel provides a targeted resequencing solution for comprehensive evaluation of somatic variants associated with childhood and young adult cancers.
| Component | Number of Pools | Concentration | Number of Amplicons | Average Amplicon Length (bp) | Average Library Length (bp) |
|---|---|---|---|---|---|
| DNA | 2 | 4X | 3069 | 114 | 254 |
| RNA | 2 | 5X | 1701 | 122 | 262 |
Completing the AmpliSeq for Illumina Childhood Cancer Panel protocol requires the panel and the following library prep, cDNA synthesis, and index kits. The AmpliSeq for Illumina Library PLUS Kit is available in 24-, 96-, and 384-reaction configurations.
The AmpliSeq for Illumina Childhood Cancer Panel generates one DNA and one RNA library per sample. Samples can be DNA, RNA, or paired. Samples for this protocol are paired. A sample is a specimen of nucleic acid from one source, while a library is nucleic acids from a sample after preparation. A library is a unique DNA or RNA preparation that has the ends required for sequencing.
Preparing the appropriate number of samples requires the following number and combination of kits.
| Number of Samples | 24 Samples | 96 Samples | 384 Samples |
|---|---|---|---|
| Number of Libraries | 48 Libraries (24 DNA, 24 RNA) |
192 Libraries (96 DNA, 96 RNA) |
768 Libraries (384 DNA, 384 RNA) |
| AmpliSeq for Illumina Childhood Cancer Panel | 1 | 4 | 16 |
| AmpliSeq Library PLUS For Illumina | 2 24-reaction kits | 2 96-reaction kits | 2 384-reaction kits |
| AmpliSeq CD Set A (96 reaction, 96 indexes) | 1 | 2 | 8 |
| cDNA Synthesis | 1 | 1 | 4 |
Sequence AmpliSeq for Illumina Childhood Cancer Panel libraries using the following systems and reagent kits.
| System | Reagent Kit | Max # DNA Samples Only per Run |
Max # RNA Samples Only per Run |
Max # Combined* Samples per Run |
Recommended Combined* DNA:RNA Pooling Volume Ratio** |
Run Time |
|---|---|---|---|---|---|---|
| MiniSeq System | MiniSeq Mid Output Reagent Kit | 1 | 8 | 1 | 5:1 | 17 hours |
| MiniSeq High Output Reagent Kit | 5 | 25 | 4 | 5:1 | 24 hours | |
| MiSeq System | MiSeq Reagent Kit v2 | 3 | 15 | 2 | 5:1 | 24 hours |
| MiSeq Reagent Kit v3 | 5 | 25 | 4 | 5:1 | 32 hours | |
| NextSeq System | NextSeq Mid Output v2 Kit | 27 | 96 | 22 | 5:1 | 26 hours |
| NextSeq High Output v2 Kit | 83 | 96 | 48 | 5:1 | 29 hours |
* Combined means paired DNA and RNA from the same sample that generates two libraries, one from each nucleic acid and separately indexed.
** Combined DNA to RNA pooling ratio based on recommended read coverage.