Next-gen Sequencing

Question 1

Issues that affect SNP calling

Answer 1

coverage (more coverage= more confidence), error rates, poloidy (need enough coverage to determine heterozygosity)

Question 2

When is exome sequencing useful?

Answer 2

In mendelian disorders.

Question 3

Illumina vs. pacific biosciences

Answer 3

Both optical
Pacific has longer reads
Illumina sequences way more templates
Illumina also has lower error rates

Question 4

Key idea behind next-gen

Answer 4

PARALLEL observation of spatially separated sequencing reactions

Question 5

Microarray

Answer 5

Rely on hybridization
Background hybridization & signal saturation limit the measurement of high and low abundance transcripts
Do not provide sequence-level information, can miss mutations, modifications and splice forms
Search space limited to probes on the array

Question 6

Next-Gen

Answer 6

Direct sequencing of cDNA products
Can identify mutations and modifications
Direct measurement of splice form abundance
No limit on search space
Dynamic range ~ depth of sequencing
Need high coverage to quantitate low-abundance transcripts

Question 7

To see cancer fusions

Answer 7

They are expressed, so sequence with next-gen