Sureselect xt human all exon 50 mb kit

Genomic DNA was extracted from the peripheral blood of the participants with a QIAamp DNA Blood Mini Kit (QIAGEN) following the manufacturer’s specifications. DNA fragments were enriched, and WES was performed using the Agilent SureSelectXT Human All Exon 50 Mb kit.

DNA of a single affected individual (IV-2) was analyzed by WES on the HiSeq2000 platform (Illumina, Inc., San Diego, CA). Sure-Select XT Human All Exon 50 Mb kit (version 5; Agilent Technologies, CA) was employed to perform the exomes enrichment while, Burrows-Wheeler Aligner (BWA v 0.7.5) was employed to align all the reads against the human assembly hg19 (GRCh37). However, Software Asset Management (SAM) tools (v0.1.18) [11 (link)], PINDEL (v0.2.4t) [12 (link)], and Exome Depth (v1.0.0) [13 (link)], were used for variant calling. Subsequently, all the variants obtained after filtering were then analyzed and subjected to Sanger sequencing for segregation within the family.

Five candidate genes, GALNT2, DCHS2, ENTPD8, PNPLA7, and FBXW7, with potential involvement in IDR drug resistance were identified by CGH array analysis. These five gene exomes were enriched and indexed using the SureSelect XT Human All Exon 50 Mb kit (Agilent, Santa Clara, CA, USA). The samples were sequenced as 100-bp paired-end runs on a HiSeq2000 system (Illumina, San Diego, CA, USA).
Next, sequences around the stop codon of mutated GALNT2 were examined. Total DNA was isolated from the MOLT-3 and MOLT-3/IDR cell lines and used in the following PCR mix: 5 μL of 2× KOD FX Neo Buffer, 1 µL of dNTPs (2 mM), 1 µL of each GALNT2 primer (2.5 µM), 1.9 µL of milliQ water (Merck Millipore Corporation, Darmstadt, Germany), 0.1 µL of KOD FX Neo (Toyobo, Osaka, Japan), and 1 µL of template DNA (100 ng/µL) from MOLT-3 or MOLT-3/IDR. The PCR amplification conditions were as follows: a denaturation step at 94 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 66 °C for 20 s, and extension at 68 °C for 20 s, and a final extension step at 68 °C for 7 min. All PCR products were fractionated by electrophoresis on 1.5% agarose gels containing TBE at 50 V for 60 min. PCR products of the appropriate sizes were purified, processed by EXOSAP-IT, and sequenced (ABI 3500xL) using PCR primers. GALNT2 DNA sequence data were assembled using ATCG software.

The target gene panel (TGP) analysis was performed on cases who had not received a diagnosis from either karyotype or CMA and their biological parents. Isolated DNA samples were sent for exome sequencing followed by TGP analysis according to De Ligt and colleagues [16 (link)]. The intellectual disability gene panel encompassing 1,252 genes (version DG-2.16) available at Diagnostics Nijmegen Laboratory and based on the last genome build reference available (hg19/GRCh37) and exome wide CNV analysis. Preparation and enrichment of genomic DNA were done using Agilent SureSelectXT Human All Exon 50Mb kit. Exome sequencing was performed on the HiSeq 2500 System platform (Illumina, USA), providing 20x coverage of > 94% of targeted bases. The variants found were classified according to the Association of Clinical Genetic Laboratory Diagnostics (VKGL) and Association for Clinical Genetic Science (ACGS) [17 ].

DNA was extracted from whole blood samples collected from the 27 patients. Targeted gene sequencing was performed on DNA samples enriched using the SureSelectXT Human all Exon 50 Mb kit (Agilent, Santa Clara, CA, USA) on the Illumina HiSeq sequencing system (Illumina, Inc., San Diego, CA, USA) with 100-bp paired end reads. Sequencing alignment and variant calling were performed using NextGENe software (SoftGenetics, State College, PA, USA).