DNA sequencing Flashcards
key idea underlying next-generation DNA sequencing
PARALLEL observation of spatially separated sequencing reactions
~1 billion 50 nt DNA sequences generated
Types of next-generation DNA sequencers
Optical (Illumina)
- read length 50-250 bases “short reads”
- # templates sequenced 10-250 million
- error rates Low (~0.1%)
- cost/time $1000 for 2 day run
Optical {Pacific Biosciences)
- read length 2500 bases “long reads”
- # templates sequenced 100 thousand
- error rates High (15%)
- cost/time $100; 30 minute run
Microarrays
relay 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 limiited to probes on the array
Next-gen sequencing
Direct sequencing of cDNA products
Can identify mutations and modifications
Direct measurement of splice from abundance
No limit on search space
Dynamic range ~ depth of sequencing
Need high coverage to quantitate low-abundance transcripts
what is the goal of mutation detection
Detect SNV (single nucleotide variants) in a genomic DNA sample
use repeated reads to compare to a reference
what are the issues affecting SNP calling
Coverage (more coverage = more confidence)
Error rates: library quality & sequencing platform-specific
Ploidy: need enough coverage to determine heterozygosity
What percentage of genome codes for proteins?
about 1%
What is exome sequencing
Build a sequencing library from noncontiguous parts of a genome, i.e., the protein coding parts minus all of the introns and other non-coding portions
How many mutations from one generation to the next
~60 de novo mutations per person per generation
how to identify chromosomal translocations using next-generation DNA
key idea: oncogenic gene fusions are expressed and provide a selective advantage (i.e., the locus is abundant in a population of cells)
examples of oncogenic fusions
BCR-ABL (Philadelphia Chromosome)
EWS-FLI1 (xxx sarcoma)
What is metagenomics
DNA of all the buggies all over your skin, in mucosa, gut
What influences extended genotype
obesity, sex, body polarity,…
What is a VUS
genetic Variant of Unknown Significance, i.e., a mutation that without an associated function
If BRCA1/2 has a VUS, do you treat somehow?
