Interpretation Next Generation Sequencing (Bench Clinic) Siamak Saber (M.D, Ph.D) Russian Academy of Medical Science

Introduction Focuses on the part of the genome we understand best, the exons of the genes Exons comprise 1% of the genome ~85% of all known disease causing mutations are located on exons

Next-generation sequencing (NGS) allows for the fast generation of thousands to millions of base pairs of DNA sequence of an individual patient. Today, the NGS method has dominated sequencing space In genomic research, and quickly entered clinical practice.

Compared with Sanger sequencing and NGS Speed Cost Accuracy Amount of samples Number of targets

Target Exome Sequencing With targeted sequencing, a subset of genes or regions of the genome are isolated and sequenced. Targeted approaches using next-generation sequencing (NGS) allow researchers to focus time, expenses, and data analysis on specific areas of interest. Such targeted analysis can include the exome (the protein-coding portion of the genome), specific genes of interest (custom content), targets within genes, or mitochondrial DNA.

Example (T.E.S) 13 genes responsible for LQT syndromes disorder. KCNQ1: 19 Ex. KCNH2: 16 SCN5A: 28 . 13*20= 260 Ex. 4 genes add, from last 2015 to now.

Advantages of Targeted sequencing 1) Focuses on regions of interest, generating a smaller, more manageable data set 2) Reduces sequencing costs and data analysis burdens 3) Reduces turnaround time compared to broader approaches 4) Enables deep sequencing at high coverage levels for rare variant identification

Whole Exome Sequencing (W.E.S)

Panel vs Exome

  Sequencing depth (also known as read depth) describes the number of times that a given nucleotide in the genome has been read in an experiment.

Target Exome Sequencing Coverage For example, the National Human Genome Research Institute of the US National Institutes of Health published a paper in 2011 pointing out that in their analysis of a single sample, almost 30% of the variants in the exome of that sample were missed with a 30x coverage of that sample as a whole-genome sequencing experiment.

HGVS Classification Pathogenic This variant directly contributes to the development of disease. In the case of recessive or X-linked conditions, a single pathogenic variant may not be sufficient to cause disease on its own. Likely pathogenic This variant is very likely to contribute to the development of disease however, the scientific evidence is currently insufficient to prove this conclusively. Additional evidence is expected to confirm this assertion of pathogenicity, Uncertain significance There is not enough information at this time to support a more definitive classification of this variant. Likely benign This variant is not expected to have a major effect on disease Benign This variant does not cause disease. ECGI Aug-2016

Columns Description Chr Chromosome Number Position SNP position Ref Reference Allele Alt Alternative Allele Func.refGene This column indicates whether the SNP falls in the exonic, intronic or intergenic region. Gene.refGene This column indicates the gene name GeneDetail.refGene This column show the intergenic distance ExonicFunc.refGene This column indicates SNP type AAChange.refGene This column indicates the amino acid change snp138 This column specifies the rsId for the SNP clinvar_20150305 This column indicates the clinical significance of the mutation gwasCatalog This column indicates SNPs identified by published GWAS studies 1000g2014oct This column contains alternative allele frequency data from 1000 genome project for autosomes and sex chromosomes SIFT_pred This column indicates function prediction of the SNP ( D: Deleterious (sift<=0.05); T: tolerated (sift>0.05) Polyphen2_ This column indicates function prediction of the SNP(D:Probably damaging (>=0.909),P:possibly damaging (0.447<=pp2_hdiv<=0.909);B: benign (pp2_hdiv<=0.446) ) MutationTaster_pred This column indicates function prediction of the SNP(A"("disease_causing_automatic");"D"("disease_causing");"N"("polymorphism"); "P"("polymorphism_automatic") FILTER This column indicates flteration categories for SNPs.LowQual=Low quality variant, PASS=SNPs passing the quality filteration.

POLYPHEN-2 This mutation is predicted to be PROBABLY DAMAGING with a score of 0.976 (sensitivity: 0.76; specificity: 0.96)

However, most mutations identified by NGS are variants of unknown clinical significance (VOUS).  Example: University of Chicago (2014) 1) Early Infantile Epileptic Encephalopathy (21 genes) Abnormal (10%), VOUS (35%), Normal (55%) 2) Infantile and Childhood Epilepsy (73 genes) Abnormal (9%), VOUS (82%), Normal (9%) 3) Limb Girdle Muscular Dystrophy (24 genes) Abnormal (38%), VOUS (62%),