A targeted, multiplex PCR primer panel was designed using the custom Ion AmpliSeq Designer v5.4.1 (Life Technologies, Carlsbad, CA, USA). The primer panel covered 42.21 kb of target regions that included potential mutations identified from MuTect1 software. Additionally, we also included two reported highly recurrent mutations in the NOTCH1 gene: (1) chr9: 139390648 (2) chr9: 139390152 (non-coding region); and mutations in the TP53, ATM, MYD88 and POT1 genes (coding region only). Design coverage of the intended targets was 98.9%. Average amplicon size of the 228 amplicons in the design was 242 bp. Sample DNA (30ng) was amplified using this custom AmpliSeq primer pool, and barcoded libraries were prepared for each sample following the manufacturer’s Ion AmpliSeq Library Preparation protocol (Life Technologies, Carlsbad, CA, USA). After library preparation, up to 96 libraries were pooled, templated and sequenced on the Proton Sequencer using the Ion P1 200bp Kit and the P1 chip per manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA).
Ion ampliseq library preparation protocol
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion AmpliSeq Library Preparation protocol is a laboratory procedure designed to generate amplicon libraries for sequencing on Ion Torrent platforms. It is a targeted sequencing approach that enables the amplification and preparation of specific genomic regions of interest.
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5 protocols using ion ampliseq library preparation protocol
1
Targeted multiplex PCR for mutation detection
2
Comprehensive BRCA1/2 Gene Sequencing
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Complete sequencing of the exons of BRCA1 and BRCA2 genes was performed in the Ion Torrent Personal Genome Machine (PGM) Sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Sequence reads were mapped to the hg19 reference genome and used to generate BAM files; variants were predicted using the both Torrent Suit Variant Caller (TSVC, Thermo Fisher Scientific, Waltham, MA, USA) and the Genome Analysis Toolkit (GATK, Intel Corporation, Santa Clara, CA, USA).
The sequencing process started with 30 ng of DNA, which was processed according to the standard protocol Multiplex Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies, Carlsbad, CA, USA). The panel amplifies 167 amplicons that cover about 16.3 kb and result in coverage between 98–100% of the coding regions of both genes. The libraries were designed according to the manufacturer by the platform Ion AmpliSeq Library Preparation Protocol (Life Technologies, Carlsbad, CA, USA). Each sample was labeled individually and added to the emulsion and subsequently sequenced using a P1 chip. Each run of that protocol produced about 10 Gb of information and each sample had an average depth of 500X [27 (link)].
The sequencing process started with 30 ng of DNA, which was processed according to the standard protocol Multiplex Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies, Carlsbad, CA, USA). The panel amplifies 167 amplicons that cover about 16.3 kb and result in coverage between 98–100% of the coding regions of both genes. The libraries were designed according to the manufacturer by the platform Ion AmpliSeq Library Preparation Protocol (Life Technologies, Carlsbad, CA, USA). Each sample was labeled individually and added to the emulsion and subsequently sequenced using a P1 chip. Each run of that protocol produced about 10 Gb of information and each sample had an average depth of 500X [27 (link)].
3
Targeted Multiplex PCR Panel for Cancer Genes
4
Validating Exome Sequencing Findings in TPP1
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Validation of exome sequencing findings in the NCI-275 family was performed by sequencing the entire genomic locus surrounding TPP1 (chr16: 67690393–67695778). A targeted, multiplex PCR primer panel (Supplemental Table S4) was designed using the custom Ion AmpliSeq Designer version 3.0 (Life Technologies). The primer panel covered 95.85% of the ACD gene, including introns and upstream and downstream sequence. Average amplicon size was 224 bp. DNA was amplified using this custom AmpliSeq primer pool, and libraries were prepared following the manufacturer’s Ion AmpliSeq library preparation protocol (Life Technologies). Individual samples were barcoded, pooled, templated, and sequenced on the Ion Torrent PGM Sequencer using the Ion PGM Template OT2 200 and Ion PGM Sequencing 200v2 kits per the manufacturer’s instructions. Mean read length after sequencing was 140 bp.
5
Targeted, Multiplex PCR for Variant Detection
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A targeted, multiplex PCR primer panel was designed using the custom Ion Ampliseq Designer v3.0 (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA). The primer panel covered 11 kb of sequence that includes the specific variants of interest in the MSH6 and DPH2 loci. Each site was 100% covered in the design. Average amplicon size was 225 bp. Sample DNA was amplified using this custom Ampliseq primer pool, and libraries were prepared following the manufacturer's Ion Ampliseq Library Preparation protocol (Life Technologies, Carlsbad, CA, USA). Individual samples were barcoded, pooled, templated, and sequenced on the Ion Torrent PGM Sequencer using the Ion PGM Template OT2 200 and Ion PGM Sequencing 200v2 kits per manufacturer's instructions. Mean read length after sequencing was 159 bp.
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