Introduction. HaloPlex panels are panels that concentrate on targeted gene sets for definite applications. Panels with preselected content or panels that are custom designed can be purchased for subsequent targeted resequencing on Next-generation sequencing platforms. Predesigned targeted gene panels consist of important genes/gene regions responsible for a disease or phenotype selected from publications and expert guidance. The panels are available for investigation of different diseases such as cancer, inherited disorders, cardiac conditions and autism. In Custom targeted gene sequencing researchers can target regions of the genome related to their definite research interests.
Methods and materials. SureDesign Online design software (Agilent Technologies) was used to create a custom HaloPlex cardiopanel for Targeted resequencing of 96 genes associated with cardiac arrhythmias using Targeted enrichment approach. The final design was done using Human Genome version 19, GRCh 37, February 2009 for Illumina platform, length of the reads is 150 bp. Candidate Gene Libraries were prepared for 48 DNA samples obtained from patients with cardiac arrhythmias using HaloPlex Custom Panel Tier 1 kit, Agilent Technologies (ILMFST, p/П G9901C) according to the manufacturer’s protocol «HaloPlex Target Enrichment System for Illumina Sequencing», version D.3. December 2012. The HaloPlex protocol is optimized for digestion of 225 ng of gDNA. Enrichment Control DNA (ECD) that is provided with the kit was used as a control.
Results. The Advanced wizard option was used to develop a design from multiple probe groups. In our case there were 3 probe groups that needed to be included in one cardiopanel. Two probe groups were already in two predesigned targeted gene panels of Agilent Technologies - HaloPlex cardiomyopathy and HaloPlex Arrhythmia, that consisted of 34 and 21 genes responsible for inherited forms of cardiomyopathy and arrhythmias, respectively. The third probe group that had to include 41 genes associated with different arrhythmogenic syndromes based on literature search was made by us. After the final panel was created, the panel was downloaded and our targets were reviewed using the UCSC Genome Browser where needed. The size of the target region was 463.767 kbp. 19958 amplicons were generated by the software to cover target regions. 99,46% of the targets were covered successfully. To prepare the Candidate Gene Library firstly, DNA samples were cut into different fragments by restriction enzymes, and denatured. Then, the probe library was hybridized to both ends of the targeted fragments guiding the targeted fragments to create circular DNA molecules. 48 different sample index sequences were added to 48 DNA samples, the probe had a method-specific sequencing motif in this step. Further, the HaloPlex probes were biotinylated and the targeted fragments were recaptured with magnetic streptavidin beads. The DNA circular molecules were joined together in a ligation reaction. Finally, targeted fragments were amplified with PCR, producing an enriched and barcoded amplification product that are ready for sequencing.
Conclusion. In conclusion, a custom HaloPlex cardiopanel for Targeted resequencing of 96 genes associated with cardiac arrhythmias was created using SureDesign Online design software (Agilent Technologies). Candidate gene libraries were prepared for 48 DNA samples in parallel using the HaloPlex cardiopanel. Further, all 48 DNA libraries will be sequenced on Illumina HiSeq2000 sequencer.