Ion AmpliSeq™ Library Kit 2.0

PCR Multiplex PCR - Mammalian DNA

Experiment
PCR Multiplex PCR - Mammalian DNA
Product
Ion AmpliSeq™ Library Kit 2.0 from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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.

Full paper   Login or join for free to view the full paper.

Reviews

Ion AmpliSeq™ Library Kit 2.0 from Thermo Fisher Scientific has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Discussion

5 years ago

Author: R. Verma India

Multiplex PCR and qPCR

I have been doing multiplex PCR and qPCR separately. Is there a way to combine them in a single reaction using Sybr Green?

Discussion

5 years ago

Author: Roland Lafontaine France

My multiplex PCR has decreased in sensitivity

Hello there! My multiplex PCR has decreased in sensitivity even though I have not changed any of the parameters. The materials are in good condition and the PCR machine is well calibrated. I have tested multiple samples but one of my targets has decreased in sensitivity.

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Multiplex PCR - Mammalian DNA using Ion AmpliSeq™ Library Kit 2.0 from Thermo Fisher Scientific.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Ion AmpliSeq™ Library Kit 2.0 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Multiplex PCR - Mammalian DNA using Ion AmpliSeq™ Library Kit 2.0 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms