Download Clinical Next Generation Sequencing (From Bench to Clinitions)

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
1. This variant directly contributes to the development of disease.
2. 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
1. This variant is very likely to contribute to the development of
disease however, the scientific evidence is currently insufficient to
prove this conclusively.
2. 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.
26
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%),
Pathogenic
18%
Benign
37%
VOUS
41%
Likely
benign
3%
Likely
pathogenic
1%