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The adapters used in this assay are optimized for the AmpliSeq workflow. Nextera or TruSeq Adapters are not compatible with this assay.
For rare clones, the recommended input amount is 1000 ng.
It is possible to run 3 different AmpliSeq for Illumina designs each with barcodes on the same sequencing run. However, your target amplicon size and required coverage must be achieved in a single run.
A 2×151 bp paired-end read is recommended.
The number of samples that can be sequenced is highly dependent on the T cell clone diversity of the sample.
Yes, Illumina recommends spiking in PhiX for low diversity samples such as Jurkat. Follow the appropriate denature and dilute guide (≥5%) for your sequencing system.
You can manipulate coverage by increasing sequencing throughput (eg, a larger flow cell output or sequencing platform) or reducing the number of samples pooled per run.
Illumina recommends using the third party MiXCr Labs app available in BaseSpace Sequence Hub.
For an example data from an experiment, go the Public Data page in BaseSpace.
On-target bases shows the percentage of total sequenced bases that map to target regions in the reference genome. This metric reflects the percentage of bases from amplicons that a) were designed, synthesized, and pooled and b) generated sequence data mapping to the target regions.
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