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Next Generation
Sequencing
A paradigm shift practice with
great opportunity and challenge
Next Generation Sequencing
• What can NGS do:
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Detects nucleotide substitution
Detects indel
Detects CNV
Detects translocation
Detects inversion
Detects methylation
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Next Generation Sequencing
• What are involved to do NGS:
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Panel design and target capture
Bar-coding for multiplexing
Sequencing (choices of platform)
Informatics infrastructure
Sequence data analysis pipelines
Data (Variant interpretation)
Examples of NGS platforms
100Mb $500 100bp/reads
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
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Clinically actionable--YES
Clinically valid but not actionable--YES/NO
Unknown or no clinical significance—NO
Presumed benign/known benign--NO
Counseling
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Pre-test
Post-test
Update
Who gets the test? Who should know the
results? (part of the results or all)
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448 severe recessive
childhood diseases
7717 regions from 437
target genes
93% of target nucleotide
with >20X coverage
~95% sensitivity
~100% specificity
104 samples tested
Average carrier burden
2.8 (0-7)