Search

Purpose Hearing loss (HL) is the most common sensory disorder in children. Prompt molecular diagnosis may guide screening and management; especially in syndromic cases when HL is the single presenting feature. Exome sequencing (ES)is an appealing diagnostic tool for HL as the genetic causes are highly heterogeneous. Methods ES was performed on a prospective cohort of 43 probands with HL. Sequence data were analyzed for primary and secondary findings. Capture and coverage analysis was performed for genes and variants associated with HL. Results The diagnostic rate using ES was 37.2% compared to 15.8% with clinical HL panel. Secondary findings were discovered in three patients. For 247 genes associated with HL, 94.7% of the exons were targeted for capture and 81.7% of these exons were covered at 20× or greater. Further analysis of 454 randomly selected HL-associated variants showed 89% were targeted for capture and 75% were covered at a read depth of at least 20×. Conclusion ES has an improved yield to clinical testing and may capture diagnoses not initially considered due to subtle clinical phenotypes. Technical challenges were identified, including inadequate capture and coverage of HL genes. Additional considerations of ES include secondary findings, cost, and turnaround time.

PURPOSE: As massively parallel sequencing is increasingly being used for clinical decision-making, it has become critical to understand parameters that affect sequencing quality and to establish methods for measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and have established a set of standards for variant calling in clinical sequencing applications. METHODS: To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCid) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in ten projects from sequencing centers in the NHGRI/NCI Clinical Sequencing Exploratory Research (CSER) Consortium. RESULTS: This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers. CONCLUSIONS: This report provides a process roadmap for clinical sequencing centers looking to perform similar analyses on their data.

BACKGROUND AND AIM: Therapeutic goals for Crohn's disease (CD) have evolved from a mere control of symptoms to the concept of deep remission (DR), including clinical and biomarker remission and mucosal healing (MH). Biologic therapy with anti-TNFα is effective in achieving MH. Yet, CD is a transmural disease, characterized by a progressive bowel damage leading to complications. This is the first pediatric study prospectively evaluating the efficacy of anti-TNFα therapy in inducing clinical remission, MH and transmural healing (TH) in ileal CD. METHODS: Pediatric patients (pts) with ileal CD starting biological therapy with Infliximab or Adalimumab were prospectively enrolled. All pts were Naive to biologics. Clinical activity (Pediatric Crohn's Disease Activity Index, PCDAI), laboratory tests (CRP, ESR), endoscopic activity (simple endoscopic score, SES-CD) and transmural disease assessed by small intestine contrast ultrasonography (SICUS) were evaluated before starting (T0) and after 9-12 months of therapy (T1). Complete MH was defined as a SES-CD of 0-1, partial MH as 50% decrease vs T0. At US the evaluated parameters were: extension of disease (cm), bowel wall thickness >3 mm (BWT), bowel wall vascularity, stratification of the BW (BWS), presence of stricture, fistulae and abscess. Wilcoxon signed rank test was used for pair comparison (T1-T0). RESULTS: 26 pts (mean age 13.3 ± 4, 16 males) were included. The mean PCDAI, ileal SES-CD, CRP, ESR, BWT and disease extension values significantly decreased at T1 (table). Increased bowel wall vascularity was present in 80% of pts at T0 and in 24% at T1 (p

Aim Capture and sequence by NGS the 4Mb of the MHC and derive genotypes of 33 MHC genes (HLA and non-HLAs) using Omixon Target HLA Typing software. Methods We applied our Region Specific Extraction method to target and capture 4Mb of the MHC region with custom designed capture oligos on 7 samples having known HLA genotypes: 5 homozygous cell lines (COX, DBB, JVM, PGF, and QBL) and 2 heterozygous samples (mixture of PGF and COX DNA, and a standard lab control). Each sample was whole-genome amplified, prepared for sequencing using Illumina TruSeq DNA sample prep kit and size selected for DNA fragments ranging from 300-400 bp. Paired-end 2x100bp sequencing was performed on the Illumina HiSeq. Each sample was genotyped for 33 genes within the MHC by Omixon Target using all paired-end reads. Derived genotypes were compared to known genotypes from the IMGT/HLA database for the cell lines and Sanger sequencing for the heterozygous control sample. Results We sequenced 7 samples throughout the 4Mb of the MHC region. On average, each sample had 284 million reads which were analyzed with Omixon Target. The 33 genes examined were Class I HLA-A, B, C, E, F, G, H, J, K, L, and V, Class II genes HLA-DMA, DMB, DOA, DOB, DPA1, DPB1, DQA1, DQB1, DRA, DRB1, DRB3, DRB4, DRB5, Class II pseudogenes HLA-DRB2, DRB6, DRB7, DRB8, DRB9, and MHC related genes MICA, MICB, TAP1, and TAP2. Omixon Target provided genotypes for 90% of the possible alleles; the remaining 10% of alleles (no genotype) were Class II pseudogenes. The average depth across the alleles ranged from 50–3000, a small percentage of the overall reads per sample. In the cases where the true HLA genotype was known, we determined the correct allele with 93% accuracy. Conclusions Our method of capturing and sequencing the MHC coupled with Omixon Target has yielded highly accurate genotypes for 33 HLA and non-HLA, MHC genes. This work sets the stage for further detailed characterization of the MHC and derivation of clinically relevant HLA typing from MHC NGS data. Major: Omixon, Inc.: Other: Contractor. Hague: Omixon, Inc.: Other: Contractor. Berces: Omixon, Inc.: Employee; Stockholder.

Aim To sequence the full length of HLA Class I genes using single molecule real-time (SMRT ® ) sequencing technology. Methods Two samples were amplified by Long-Range PCR at HLA-A, B, C loci. Full lengths of these genes (∼3.2 kb) were generated with custom designed, barcoded primers targeting 5’UTR and 3’UTR. DNA libraries were prepared by ligating SMRTbell™ adaptors to the HLA amplicons. This produced closed-circular structures consisting of a double-stranded insert (full length HLA amplicon), and a single-stranded hairpin loop (SMRTbell™) on either end. Sequencing primer was annealed to the open/single-stranded portion of the hairpin adaptor. Streptavidin-bound DNA polymerase was added to the primer-template complex enabling the binding of the template to the bottom of nano-sized wells on the SMRT Cell. DNA synthesis of fluorescently labeled nucleotides was observed in real-time on the PacBio ® RS instrument. The pooled libraries were run on 6 SMRT Cells using either 45 or 90 minute data collection time per SMRT Cell. Individual sequence reads ⩾1,000 bases were parsed by sample-specific barcode sequence and were then aligned using BWA-SW. Alignment data were analyzed with SAM Tools and custom written code. Results Sequence and Alignment Statistics: [ Table 1 ]. Conclusions We demonstrate real-time sequencing of HLA genes from single DNA molecules. This technology offers great potential for HLA genotyping and further characterization of the MHC due to the length and phase of sequences obtained, speed, and ease of sample prep. Ranade: Pacific Biosciences Inc.: Employee. Sethuraman: Pacific Biosciences Inc.: Employee. Chin: Pacific Biosciences Inc.: Employee. Robinson: Pacific Biosciences Inc.: Employee.

Aim To evaluate newly available high-throughput sequencing technologies for HLA genotyping. Methods Two samples were amplified at HLA-A, B, C, DRB1, and DQB1. The full length of HLA Class I genes were amplified with custom designed primers from 5′UTR to 3′UTR. HLA Class II were amplified from Exon 2 to Exon 4 using primers from GenDx. A separate DNA library was prepared from each long-range PCR amplicon using platform-specific library prep kits (NEB, Ipswich, MA). For the Ion Torrent run, the pool of 10 libraries was clonally amplified by emulsion PCR and was sequenced using the 200 bp kit and a 316 chip on the Ion Torrent PGM (Life Technologies). Paired-end 2 × 150 bp sequencing of the Illumina libraries was performed on the MiSeq system. Sequence alignment utilized BWA-SW software. Further analysis was conducted using SAM tools and custom written code. Sequence analysis was performed in parallel with NGSEngine (GenDx). Results Sequencing and alignment metrics were as follows:[ Table 1 ] Consensus sequence of aligned reads was 100% accurate for both platforms. Definitive HLA genotypes were obtained without any ambiguity (alternative genotypes) and were concordant with the known alleles of these samples. Conclusions Newly developed high-throughput sequencing systems hold great promise for a new method of HLA genotyping. These new technologies are capable of eliminating all ambiguity currently encountered with existing methods. As such the advancement from high resolution to allele level HLA typing is approaching. Rozemuller: GenDx: Employee. Penning: GenDx: Employee. Mulder: GenDx: Employee.