Arrays And Next-Gen Sequencing-Lecture 9/29/21

Question 1

Small variants

Answer 1

Single nucleotide variants

- Short insertions, duplications and deletions <50 BP

Question 2

Structural variations (SVs)

Answer 2

Changes that are at least 50 bp in size

Question 3

Test in increasing level of resolution

Answer 3

Karyotype (2-3 Mb), FISH (120-400 kB), Array (250 kB), NGS (1 bp)

Question 4

Chromosomal microarray (CMA)

Answer 4

Compare patient hybridization to reference, deletion=less hyb and duplication= more hyb CANNOT DETECT BALANCED TRANSLOCATION

Question 5

DiGeorge syndrome

Answer 5

22q11.21 deletion syndrome, VCF syndrome, cleft palate, congenital heart defect, developmental delay, kidney anomalies, frequent infection

Question 6

Normal 2N result for alleles

Answer 6

3 bands

Question 7

Allele track for 3N

Answer 7

4 bands

Question 8

Allele track for a deletion (1N)

Answer 8

2 bands

Question 9

Clinical applications of microarrays (4)

Answer 9

• Assessment in small deletions in seemingly balanced rearrangements found on karyotype
• Developmental delay, autism
• Dysmorphic features
• Rare disease diagnostics
• Molecular characteristics of tumors

Question 10

Limitations of CMA (3)

Answer 10

• Does not identify mechanism of rearrangement
• Balanced translocations or inversions will not be detected
• May not detect low level mosaicism

Question 11

DNA sequencing examples

Answer 11

Sanger sequencing

Next gen sequencing

Question 12

Sanger sequence method

Answer 12

Fluorescent ddNTPs added and detected when the chain stops

Question 13

Applications of Next generation sequencing (3)

Answer 13

• Gene panels
• Whole exome sequencing
• Whole genome sequencing

Question 14

When to use WES (3)

Answer 14

• When other tests fail to yield diagnosis
• Long list of differential diagnoses
• Atypical presentation of genetic disorder

Question 15

Primary findings from WES

Answer 15

Medically relevant variants in disease genes known to be related to phenotype

Question 16

Secondary findings of WES

Answer 16

Medically variants in disease genes not associated with the patients phenotype
Surprises: Sex chromosome mismatch, non-paternity, etc

Question 17

Variant detection gap

Answer 17

Between CMA and NGS, about 10-25 KB

Question 18

Epigenetic changes

Answer 18

Histone modification, Nucleosome positioning, methylation-> effects on gene expression that are not alterations in the DNA sequence itself HERITABLE