Session 5 - Sequencing, Genotyping Sequencing, and Microarray

What was the goal of ENCODE?

to identify all functional elements in the human and mouse genomes

What was the goal of the NIH Roadmap Epigenomics Mapping Consortium?

to systematically characterize epigenomic landscapes in primary human tissues and cells

What is the goal of the 4D Nucleome program?

study the three-dimensional organization of the nucleus in space and time

What was the goal of the HapMap project?

develop a haplotype map of the human genome, to describe the common patterns of human genetic variation

What was the goal of the Cancer Genome Atlas?

to molecularly characterize over 20,000 primary cancer and matched normal samples spanning 33 cancer types

What was the goal of the Genotype-Tissue Expression Project?

to study human gene expression and regulation and its relationship to genetic variation in 54 tissues of ~1000 individuals

What was the goal of the human cell atlas?

to create a comprehensive reference maps of all human cells

What was the goal of the UK biobank?

to investigate the contributions of genetic and environmental factors to disease

What are the parts of the Precision Medicine Initiative in US?

All of Us Research Program, Million Veteran Program, precisionFDA, Big Data Science Initiative

What is the Telomere-Telomere consortium?

an open, community-based effort to generate the first complete assembly of a human genomeW

What is the goal of the Human Pangenome Project?

create a genome reference that is representative of human genetic diversity, over 300 high quality haplotype-resolved human genomes will be produced

What is first-generation sequencing?

Sanger sequencing

What did Sanger determine?

the complete amino acid sequence of the two polypeptide chains of bovine insulin by 1953

What did Sanger and colleagues introduce in 1977?

the dideoxy chain-termination method

What is the difference between dNTP and ddNTP?

ddNTP only has an H at the 3’ carbon while dNTP has an OH

What is the purpose of ddNTPs in Sanger sequencing?

chain-elongating inhibitors of DNA polymerase, labelled with four fluorescent dyes

What happens during Sanger sequencing?

during elongation along the DNA template, ddNTPs are randomly inserted in place of the corresponding dNTP, terminating elongation

What are the features of Sanger sequencing?

electrophoretic sequencing, high quality, long read length (800bp), poor quality in the first 15-40 bases due to primer binding and deteriorating quality after 700 bases, error prone in regions containing stretches of a single nucleotide

What is second-generation sequencing?

massively parallel sequencing/next generation sequencing

What are the key features of second generation sequencing?

multiplexing, parallelization, high-throughput, short-read length (35-250 bp), relatively high quality, cost-effective

What is a flow-cell?

a glass slide containing small fluidic channels and coated with short oligonucleotides

Does second generation need to run a gel?

no

What is sequencing by synthesis?

using DNA polymerase to synthesize the complementary strand of the DNA template, and determining the sequence of nucleotides incorporated in the process

What is a summary of Illumina?

bridge amplification + sequencing by synthesis with reversible dye-terminators

What are the applications of second-generation sequencing technology?

de novo genome assembly, genome resequencing, RNA sequencing, ChIP-sequencing, metagenome/microbiome sequencing

What is genome resequencing?

mapping sequence reads to the reference genome to identify genetic variants

What are the characteristics of targeted sequencing?

specific regions, 500x coverage, identifying all kinds of variants in specific regions, most cost effective

What does RNA sequencing do?

detects levels of gene expression, identifies new transcripts

What is ChIP-seq?

chromatin immunoprecipitation to “pull down” DNA fragments protected by a specific protein

What is a metagenome?

the collective genomes of the microorganisms that reside in an environmental nicheW

What does metagenome/microbiome sequencing allow?

identify new species, investigate biological phenomena

What is the major limitation of microarray?

only focus on pre-selected genetic variants or genes

What is a DNA microarray?

a collection of microscopic DNA spots attached to a solid surface

What does a genotyping array do?

determine genotype at pre-selected genomic positions

What does a gene expression array do?

determine express levels of pre-selected genes

What do GWAS usually apply?

genotyping arrays to determine genotypes at 500K - 1M genomic positions

What is third-generation sequencing?

real-time, single molecule sequencing

What are the key features of third generation sequencing?

amplification-free, very long read length, low throughout, less parallelization, less data output, high error rateW

What is PacBio sequencing?

a zero mode waveguide, a hole less than half the wavelength of light, limits fluorescent excitation to a tiny volume within which a single polymerase and its template reside

What is nanopore sequencing?

single-stranded RNA or DNA could be driven across a lipid bilayer through nano-scale ion channels by electrophoresis, passage through the channel blocks ion flow, decreasing the current for a length of time proportional to the length of the nucleic acid