Ion AmpliSeq™ Library Kit 2.0

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

NGS was performed using AmpliSeq Cancer Hotspot Panel (v. 2) for targeted multigene amplification as described previously.22,24 Briefly, we used Ion AmpliSeq Library Kit 2.0 for library preparation, Ion OneTouch 200 Template Kit v2 DL and Ion OneTouch Instrument for emulsion PCR and template preparation, and Ion PGM 200 Sequencing Kit with Ion 318 Chip and Personal Genome Machine as the sequencing platform (Life Technologies, Carlsbad, CA). The DNA input was up to 30 ng measured by Qubit 20 Fluorometer (Life Technologies). Up to 8 specimens were barcoded using Ion Xpress Barcode Adapters (Life Technologies) for each Ion 318 chip. One to three controls (nontemplate control, a normal peripheral blood control from a male and/or positive control specimens) were included in each chip. Sequencing data of the targeted genes was analyzed using Torrent Suite (Life Technologies). Mutations were identified and annotated through both Torrent Variant Caller and direct visual inspection of the binary sequence alignment/map (BAM) file on the Broad Institute’s Integrative Genomics ViewerIGV.25 During our validation of this NGS assay, a background noise threshold of 2% was chosen for single nucleotide variations.22 Provided sufficient DNA input, the limit of detection is dictated by the depth of coverage (or number of sequencing reads) in the target genomic region. Approximately 150 and 500 reads are needed to detect a heterozygous mutation at a 99% confidence in a specimen with 20% and 10% tumor cellularity, respectively.

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Manufacturer protocol

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