Questions & Answers

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Protocol

Does this assay use the standard Nextera or TruSeq Adapters?

The adapters used in this assay are optimized for the AmpliSeq workflow. Nextera or TruSeq Adapters are not compatible with this assay.

What is required to purchase from Illumina?
Can I perform two or three different amplifications and then pool them before going into library prep?

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.

Sequencing

What read length is recommended for sequencing?

A 2×151 bp paired-end read is recommended.  

How many samples can be sequenced at a time?
Experimentally, how does one manipulate coverage? For example, if I want 100 X, then later I want 500X, what must I change?

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.

Analysis

What tools are offered for data analysis?

The DNA Amplicon analysis workflow can be used to perform alignment and variant calling.  Local Run Manager and BaseSpace Sequence Hub have apps available for analysis. The DNA Amplicon Analysis App is available on BaseSpace Sequence Hub. Further analysis can be performed on any variant calls using BaseSpace Variant Interpreter.  Local Run Manager has a similar DNA Amplicon Analysis Module which utilizes the same workflow and algorithm as the BaseSpace Sequence Hub Apps. 

What does the data look like? Is there demo data available?

For an example data from an experiment, go the Public Data page in BaseSpace.

How is the measure for on-target bases (specificity) determined?

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.