Methodology

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

DNA analysis of the PKD1 gene is complicated and challenging due to the presence of several PKD1 pseudogenes. There is high sequence similarity of exons 1 to 33 between PKD1 and its pseudogenes (Audrézet et al. 2012. PubMed ID: 22508176). We have validated Next Generation Sequencing (NGS) to reliably sequence these exons.

For the PKD1 gene, including exons 1 to 33 (homologous regions), we primarily use Next Generation Sequencing (NGS) (~96%) complimented with Sanger sequencing for low-coverage regions (~4%). For any pathogenic, likely pathogenic, and uncertain variants found in exons 1 to 33 (homologous regions) via NGS, we use long-range PCR based Sanger sequencing to confirm them. Therefore, this test provides full coverage of all coding exons of the PKD1 gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Due to homologous sequence, gene conversion events in the PKD1 gene have been reported in the literature and found at PreventionGenetics. Our internal data suggested gene conversions are rare (<0.5%) in PKD1. These events have been found by long-range PCR based Sanger sequencing, but not by NGS only. Therefore, Sanger sequencing for exons 1 to 33 (homologous regions) of PKD1 may also be ordered.

Regarding copy number variants (CNVs) analysis, because of the paucity of CNVs and the complicated nature of sequence in PKD1, CNV analysis for this gene can be performed via the multiplex ligation-dependent amplification (MLPA) assay with limited increased sensitivity (compared to Next-Gen sequencing CNV analysis), and can be ordered separately (Test #2058).

This panel provides 100% coverage of all coding exons of the genes listed plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).