​The Center for Rare / Orphan Diseases offers the full workflow of analysis of whole exome and whole genome sequencing, as well as other NGS analysis workflows which may be applied during further investigation of disease processes, such as RNA-Seq (mRNA expression), ChIP-Seq, DNAse-seq (regulation of expression), miRNA expression profiling and more.

NGS sequencing of human samples may be preceded by genotyping using SNP and CNV DNA microarrays. This data will be preprocessed by standard tools and then candidate regions will be determined by genetic linkage mapping or through homozygosity mapping. The achieved intervals will help limit the regions considered in the subsequent NGS data analysis.
 
A typical workflow for discovering SNPs and small indels in whole exome and whole genome sequencing is composed of the following steps:
  1. Extensive quality assurance of the sequencing reads
  2. Alignment of the reads to the reference human genome
  3. Removal of PCR duplicates
  4. Variant calling, performed jointly on the relevant set of case and control samples
  5. Variant annotation and effect prediction
  6. Variant filtering and prioritization
 
Whereas steps 1-5 are quiet standard and may be done automatically, step 6 requires much biological thinking and manual adjustments. Several criteria are typically taken into consideration when deciding on the set of variants to focus on and their order of priority:
  1. Confidence of the variant call
  2. Comparison to genes and variants already known to be involved in the disease at hand
  3. Variant frequency in the general (relevant) population and known disease associations, by comparison to population Genotyping databases and to mutation databases
  4. Comparison to genotypes of local populations (using in-house database of previously analyzed exomes/genomes) and control individuals of the study at hand
  5. Prediction of how deleterious to protein expression or function a variant may be
  6. Tissue of expression of the gene harboring a variant
  7. Presence in linkage or homozygosity interval which was previously identified
  8. The biological context of the disease phenotype and symptoms
 
Whole genome sequencing enables the extension of variant identification to regulatory DNA regions in addition to the coding regions probed in Whole exome sequencing. Whereas the initial analysis steps of Whole Genome Sequencing data are the same as for exome data, the annotation, filtering and prioritization of the identified variants need to be extended to also take into account possible regulatory functions. For this end, positions of identified variants will be overlapped with genomic regions previously annotated as involved in the regulation of gene transcription.

To help compare candidate variants with genotyped individuals from relevant Israeli populations, the exome and genome genotyping data produced at the Orphan Disease Center, will be anonymously stored at a local dedicated database. Storage will be done based on agreement of the scientist doing the research.
 
Upon completion of data analysis, a final report will be prepared with links to relevant result files, as well as documentation of the analysis in the format of "Materials and Methods" of scientific publications. In addition, a meeting with the customers will be set to discuss the results and future directions.

It should be emphasized that all the findings are on a research rather than clinical basis. Prior to clinical use, the findings should be verified in a clinical laboratory.