Computational protocol: Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

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Protocol publication

[…] This Hi-Plex assay was designed to target the PALB2 and XRCC2 genes. However, genotyping aspects of this study focus on PALB2 only, as we did not have a similar test set with genotype data for XRCC2.Sixty primer pairs targeting the protein coding and some flanking intronic and untranslated regions of PALB2 and XRCC2 are described in [] and Additional file . Dual-indexed hybrid adapter primer sets are described in Additional file . All oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA).96 individual PCR reactions (95 specimen DNAs and one no-template control) were performed in a standard skirted PCR plate, in a final volume of 50 μl, with1X Phusion® HF PCR buffer (ThermoScientific, Waltham, MA, USA), 2 units of Phusion Hot Start II High-Fidelity DNA Polymerase (ThermoScientific), 400 μM dNTPs (Bioline, London, UK), approximately 0.5 μM gene-specific primer pool (individual gene-specific primer concentrations vary and are described in []), 2.5 mM MgCl2 (ThermoScientific) and 25 ng input genomic DNA. The following steps were then applied to conduct PCR: 98°C for 1 min, 6 cycles of [98°C for 30 sec, 50°C for 1 min, 55°C for 1 min, 60°C for 1 min, 65°C for 1 min, 70°C for 1 min], addition of 2 μM each dual-indexed hybrid N50#_TSIT_A and N70#_TSIT_P adapter primers, then a further 19 cycles of [98°C for 30 sec, 50°C for 1 min, 55°C for 1 min, 60°C for 1 min, 65°C for 1 min, 70°C for 1 min], followed by incubation at 60°C for 20 min. Five μl of each reaction were pooled before subjecting the resulting barcoded library (including the 96 sub-libraries) to electrophoresis on a 2% HR-agarose gel (Life Technologies). Size selection, gel extraction and purification were performed as described previously [].The library was then sequenced on a MiSeq instrument, using the MiSeq Reagent kit v2 300 cycles (Illumina). Prior to performing the run, 3.4 μL of 100 μM sequencing primers were added to the respective read1, read2 and i7 primer reservoirs in the reagent cartridge. Sequencing primers were obtained from Integrated DNA Technologies (sequences are provided in Additional file ).Sequencing data were mapped to the entire human genome (hg19) using bowtie2-2.1.0 [] applying default parameters except for --trim5 20 --trim3 20. Bedtools v2.16.1 [] was used to compute on-target coverage. We used ROVER variant caller, a software tool developed in-house and made available at to perform automated variant calling. To be called in this application, genetic variants had to appear in i) both members of read-pairs; ii) at least 2 read-pairs; and iii) ≥ 15% of read-pairs. Homozygous variants were called when the minor allele was present in ≥85% of read-pairs. The tool also reports the number of read pairs covering each targeted amplicon. Sequencing statistics reported in this paper (on-target and coverage calculations) include both XRCC2 and PALB2, as they represent all the targeted regions. To assess the efficiency of the 60-plex assay across all 95 specimens, depth of coverage data were reported for 60 × 95 = 5,700 amplicons in total.When validation was required for a genetic variant identified by Hi-Plex but not reported in previous screens, Sanger sequencing was performed using BigDye Terminator v3.1 (Life Technologies), according to the manufacturer’s instructions. […]

Pipeline specifications

Software tools Bowtie2, BEDTools, ROVER
Application Amplicon sequencing analysis
Diseases Breast Neoplasms, Neoplasms
Chemicals Formaldehyde