Molecular Genetics Lecture 5 - DNA Sequencing I

What does DNA sequencing determine?

sequencing determines the linear order (sequence) of AGCT bases in DNA nucleotides on a chromosome

What does DNA sequencing provide?

information on gene structure and regulation and diagnosis and treatment of human diseases

what do nucleotide sequences of a gene help in

deducing the protein sequences from DNA sequences and manipulating genes to study its function

what is functional genomics?

manipulating genes to study its function

what is a genome?

complete set of DNA from the whole organism

what is genomics?

study of content, function and organization of all the genes and DNA from an organism

to synthesize DNA, DNA polymerase catalyzes the reaction between a ____ and the _____

3’ OH group of last nucleotide and the 5’ phosphate group of the next nucleotide

who developed Dideoxy DNA sequencing method?

Frederick Sanger

what is Sanger sequencing?

uses a modified nucleotide dideoxyribonucleoside triphosphate (ddNTP) which lacks 3’ OH group required for polymerizing DNA

what is chain termination?

whenever ddNTPs are added isntead of dNTPs, DNA synthesis is terminated

what are the reagents required for DNA sequencing by dideoxy method?

nucleotides, modified dideoxy nucleotides, radioactively labeled (phosphorus 32) short primer, DNA template, DNA polymerase, polyacrylamide gel electrophoresis

what (non-modified) nucleotides are used for DNA sequencing by dideoxy method?

Deoxyribo nucleoside triphosphates (dNTPs: dATP, dGTP, dCTP, dTTP)

what are the modified dideoxy nucleotides?

dideoxyribo nucleoside triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, ddTTP)

what is polyacrylamide gel electrophoresis for DNA sequencing by dideoxy method?

separates DNA fragments with one bp nucleotide difference

what reagents are in all 4 tubes of dideoxy/sanger sequencing?

DNA template, nucleotides, radiolabeled primer, DNA polymerase

what is chromatogram?

DNA sequencing results where each nucleotide base is represented with a different color

Sanger sequencing sequences ___ DNA fragment (read) in each reaction

one

What can Next generation sequencing technologies do?

sequences many millions DNA fragments (reads) at the same time (massively parallel) for cheap and fast

what is an exome?

all the exons in a genome

what is Next generation sequencing (NGS) also known as?

high throughput sequencing (HTS)

what is an example of First Generation Sequencing?

sanger sequencing

what is Second (Next) Generation Sequencing?

massively parallel sequencing

what is an example of Second (Next) Generation Sequencing?

Illumina sequencing, 454 pyrosequencing, SOLiD sequencing

what is Third Generation Sequencing?

single molecule sequencing

what is an example of Third Generation Sequencing?

Pacific Biosciences (PacBio), Oxford Nanopore

what is De novo sequencing?

Whole genome sequencing of a species (human) for the first time, no other genome information is available

what are examples of De novo sequencing?

PacBio and Oxford Nanopore Technologies

PacBio and Oxford Nanopore Technologies have ____ reads and ____ coverage for de novo sequencing?

long reads and less coverage

what is re-sequencing?

whole genome sequences are already available for the species (reference genome) and sequencing of a different individual of a species of an organisms

Illumina technology has ____ reads and ____ coverage for resequencing and used in gene expression studies

short reads, high coverage

what is genome coverage?

Is how many times reads match to the same region on a sequenced region (Reference Genome). a read can match to the same region 1x, 2x, 3x, etc

what is read length?

the length of each sequenced DNA fragment

what is read quality?

is the number of mistakes during copying nucleotides during sequencing

what is throughput?

the maximum number of nucleotides sequenced per run

what is turn around time?

time taken for sequencing

what are the important parameters to consider for NGS?

genome coverage, read length, read quality, throughput, turn around time, cost

shorter reads (Illumina technology) are ____ but can ___

hard to map to the genome but can cover genome multiple times (coverage, functional genomics)

longer reads (PacBio technology) are ___ but ____

easy to map to the genome but hard to cover multiple times (genome assembly, de novo sequencing)

what is base calling accuracy?

accuracy of sequencing

quality of sequencing is measured by _____

Phred Quality Score

PacBio has the ____ read quality

highest

Illumina has the ____ quality

low

what is the throughput and runtime for illumina?

1800 GB per run

what is the throughput and runtime for PacBio/ion torrent sequencing?

fast run time