Next Generation Sequencing

A paradigm shift practice with great opportunity and

challenge

Next Generation Sequencing

• What can NGS do:– Detects nucleotide substitution– Detects indel– Detects CNV– Detects translocation– Detects inversion– Detects methylation– …

Next Generation Sequencing

• What are involved to do NGS:– Panel design and target capture– Bar-coding for multiplexing– Sequencing (choices of platform)– Informatics infrastructure– Sequence data analysis pipelines– Data (Variant interpretation)

100Mb $500 100bp/reads

Examples of NGS platforms

Mi-Seq

1-1.5Gb $1200 150bp/run

Sanger vs. NGS

Sanger sequencing next generation sequencing

Analyzing the clinical significance of the variants

VAST ARRAY OF GENETIC DISORDERS

Chromosomal

Trisomies, aneuploidies, etc.

Metabolic

SLO, CDG, many others

Organ Specific

Cardiopathies, GI, Renal

Protein

DMD, Huntington, Immunoglobulins

Cancer

At risk mutations

And On and On

Variant categorization

• Known deleterious (condition relevant and incidental)

• Presumed deleterious• VUS (variants of undetermined

significance)• Presumed benign• Known benign

Reportability

• Clinically actionable--YES• Clinically valid but not actionable--YES/NO• Unknown or no clinical significance—NO• Presumed benign/known benign--NO

Counseling

• Pre-test• Post-test• Update• Who gets the test? Who should know

the results? (part of the results or all)• …

448 severe recessive childhood diseases

7717 regions from 437 target genes

93% of target nucleotide with >20X coverage

~95% sensitivity~100% specificity

104 samples testedAverage carrier burden 2.8 (0-7)