Computational protocol: Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism

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Protocol publication

[…] We developed a HaloPlex enrichment platform for use with Illumina HiSeq 2500 sequencing customized to identify variants in 26 nuclear genes (, including references for genes) belonging to RC and FAO disorders []. The gene panel consists of 15 nuclear genes involved in maintenance of mtDNA integrity and in MDS, five genes encoding assembly and translation factors for RC complexes, and six genes associated with FAO defects. Mutations in genes from these three groups have previously been associated with ALF, especially in infants [,]. For the DNA enrichment and library preparation, the workflow had 4 major steps; 1) DNA digestion and denaturation, 2) hybridization to probe library, 3) capture and target ligation and 4) PCR amplification of targeted fragments. In short, a total of 0.5 μg of genomic DNA was digested with restriction enzymes. After digestion, eight digestion reactions were pooled, resulting in a single DNA sample containing a 16-enzyme restriction fragment library that included both target and non-target gDNA regions. The restriction fragments were hybridized to the HaloPlex probe capture library, designed to hybridize selectively to target fragments and to direct circularization of such targeted DNA fragments, during which Illumina sequencing motifs were incorporated. Then, the biotinylated-probe/fragment hybrids were retrieved with magnetic streptavidin beads. DNA ligase was added to the capture reaction to catalyze the formation of closed-circular DNA from the circularized target DNA-HaloPlex probe hybrids. Non-target DNA in the capture reaction liquid phase remained in linear fragment form and was digested by the exonuclease activity of the provided Haloase A and B enzymes. During the Haloase B treatment, circularized target DNA was released from the streptavidin beads into the liquid phase. Then, the captured DNA was amplified and index-tagged in PCR reactions containing Illumina Primer 1.0 along with the appropriate index primer followed by a DNA purification using AMPure XP beads. The PCR products from each library was checked by the Agilent Bioanalyzer and quantitated the Qubit® 1.0 Fluorometer (Invitrogen), respectively. The samples with different indexes were pooled at equal molar concentration and sequenced as single-end reads on the HiSeq 2500 (Illumina) instrument according to the manufacturer’s protocols at the DNA sequencing core at Cincinnati Children’s Hospital Medical Center (CCHMC).Before interpretation, the data were analyzed and annotated by means of a pipeline that was developed in-house. Briefly, the output data from the HiSeq 2000 were converted from a bcl file to a FastQ file by means of Illumina Consensus Assessment of Sequence and Variation software and mapped to the reference haploid human-genome sequence (hg19) with the use of the NexGENe 2.2.3 program (Softgenetics). Variant calls, which differed from the reference sequence, were obtained with NextGENe 2.2.3. Variant prioritization was based on allele frequency, pathogenicity program predictions and mutation database searches. Alamut HT 1.1.8 and in-house scripts annotated the variants. Variants in this database with a minor allele frequency of less than 1% according to exome sequencing project database (, and within +/- 20 bp of the exon/intron boundary were retained. In addition, damaging mutations were examined by focusing on frameshift, start loss and nonsense changes, splice site mutations and missense changes with pathogenic scores as predicted by SIFT, POLYPHEN-2 and Grantham scores. Moreover, variants reported in the Human Gene Mutation Database were also prioritized. Post-filtering, promising candidate gene variants were confirmed by Sanger sequencing. […]

Pipeline specifications

Software tools NextGENe, SIFT, PolyPhen
Databases HGMD
Application WES analysis
Organisms Homo sapiens
Diseases Acidosis, Lactic, Fatty Liver, Lymphoma, Non-Hodgkin, Liver Failure, Liver Failure, Acute
Chemicals Pyruvic Acid, Lactic Acid