Mutation analysis was done by direct sequencing for the 15 exons encoding the 767 amino acid (aa) DZIP1L protein (GenBank: NM_173543.2, NP_775814; mutation numbering corresponds to the A of the ATG-translation initiation codon in exon 2). Genomic DNA from an affected individual was amplified by PCR with oligonucleotide primers complementary to flanking intronic sequences. Primers used for PCR and direct sequencing are available on request. Samples were run and analyzed on an ABI PRISM 3130 genetic analyzer (Applied Biosystems).
In addition to conventional Sanger sequencing of DZIP1L in a total of 218 patients with suspected PKD, we performed different next-generation sequencing (NGS) based approaches. First, we used PCR-based 48.48 Access Array microfluidic technology (Fluidigm™) with consecutive NGS. We applied a multiplexing approach allowing PCR-based amplification of 53 amplicons (44 exons) for 48 DNA samples simultaneously in one known and two candidate genes, including DZIP1L. A total of 96 patients with a PKD-related phenotype were analyzed. After two rounds of amplification followed by indexing of all patient-derived products with 96 different 10 bp-barcodes in a subsequent PCR, 2×150 bidirectional sequencing was performed on a MiSeq platform (Illumina™). Second, all exons and adjacent intronic boundaries of a different number of genes (dependent on the version of our customized multi-gene panel, including DZIP1L) known or hypothesized to cause PKD and other ciliopathies were targeted by a custom SeqCap EZ choice sequence capture library (NimbleGen, Madison, Wisconsin, USA) and subsequently sequenced on a Roche 454 GS FLX or an Illumina MiSeq or HiSeq platform (2×150 PE) according to the manufacturer’s protocol. A total of 1234 patients with a polycystic kidney disease phenotype (n=429) or an NPHP-related complex ciliopathy (n=805) were analyzed with an average coverage of 60-fold (GS FLX), 120-fold (MiSeq) or more than 200-fold (HiSeq), respectively. Bioinformatic analysis was performed using the Roche GS Reference Mapper™ software (v2.6), SeqPilot SeqNext moduleTM (v3.5.2, JSI medical systems, Kippenheim, Germany) as well as an in-house bioinformatic pipeline. For all approaches, potential mutations were confirmed by Sanger sequencing and shown to segregate with the phenotype. No further mutation thought to be of pathogenic relevance for the disease phenotype was present among the patients described in this manuscript. Whole-exome sequencing (WES) and mapping of reads was carried out as previously described48 (link),49 (link).
In addition to conventional Sanger sequencing of DZIP1L in a total of 218 patients with suspected PKD, we performed different next-generation sequencing (NGS) based approaches. First, we used PCR-based 48.48 Access Array microfluidic technology (Fluidigm™) with consecutive NGS. We applied a multiplexing approach allowing PCR-based amplification of 53 amplicons (44 exons) for 48 DNA samples simultaneously in one known and two candidate genes, including DZIP1L. A total of 96 patients with a PKD-related phenotype were analyzed. After two rounds of amplification followed by indexing of all patient-derived products with 96 different 10 bp-barcodes in a subsequent PCR, 2×150 bidirectional sequencing was performed on a MiSeq platform (Illumina™). Second, all exons and adjacent intronic boundaries of a different number of genes (dependent on the version of our customized multi-gene panel, including DZIP1L) known or hypothesized to cause PKD and other ciliopathies were targeted by a custom SeqCap EZ choice sequence capture library (NimbleGen, Madison, Wisconsin, USA) and subsequently sequenced on a Roche 454 GS FLX or an Illumina MiSeq or HiSeq platform (2×150 PE) according to the manufacturer’s protocol. A total of 1234 patients with a polycystic kidney disease phenotype (n=429) or an NPHP-related complex ciliopathy (n=805) were analyzed with an average coverage of 60-fold (GS FLX), 120-fold (MiSeq) or more than 200-fold (HiSeq), respectively. Bioinformatic analysis was performed using the Roche GS Reference Mapper™ software (v2.6), SeqPilot SeqNext moduleTM (v3.5.2, JSI medical systems, Kippenheim, Germany) as well as an in-house bioinformatic pipeline. For all approaches, potential mutations were confirmed by Sanger sequencing and shown to segregate with the phenotype. No further mutation thought to be of pathogenic relevance for the disease phenotype was present among the patients described in this manuscript. Whole-exome sequencing (WES) and mapping of reads was carried out as previously described48 (link),49 (link).
