RNA sequencing
- uses next generation sequencing to reveal the presence and quantity of RNA in a biological sample, can analyze the continuously changing transcriptome
- can identify alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/some SNPs and differences in gene expression in diff groups or treatments
- can interrogate other populations of RNA including total RNA, small + long non coding RNA + ribosomal RNA
- used to determine exon/intron boundaries and verify or amend previously annotates 5’ and 3’ gene boundaries
how much of total RNA is coding RNA
1%
recent advances in RNA seq include
single cell sequencing, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single molecule real time sequencing
ancient DNA challenges
- degraded by nucleases
- majority of DNA in samples derives from unrelated organisms such as bacteria that invaded after death
- majority of DNA in samples is contaminated by human DNA
- determination of authenticity requires special controls, and analysis of multiple independent extracts
comparative genomics
involves comparison of genome sequences from multiple species, or in some cases from individuals within a species
phylogentic footprinting
comparisons of genomic sequences from distantly related organisms, such as humans relative to fish, chickens, and rodents.
- especially useful to identify conserved elements (under negative selection), emphasizing the relatively rare coding and noncoding segments of the genome that remain shared even after hundreds of millions of years since species such as human and fish diverged
sanger sequencing
- template is denatured to form single strands
- extended with a polymerase in the presence of dideoxynucleotides (ddNTPs) that cause chain termination
- typical read lengths are up to 800 bps
sanger sequencing throughput
- run up to 384 samples
- 700 base reads
- up to 1 million bases per day
- human genome is 6 billion bases
- covered 7 times
- over 100 years on one machine
cycle termination sequencing (illumina) disadvantages
short read length (~150 bases) (mRNA length: a few 1000 bps)
cycle termination sequencing (illumina) advantages
- very fast
- low cost per base
- large throughput, up to 1 gigabase/experiment
- short read length. makes it appropriate for resequencing
- no need for gel electrophoresis
- high accuracy
- all 4 bases are present at each cycle, with sequential addition of dNTPs. allows homopolymers to be accurately read
illumina sequencing technology
- shear and break genomic DNA
- prepare genomic DNA (fragment genomic DNA and ligate adapters to both ends)
- attach DNA to surface
- bridge amplification
- fragments become double stranded
- denature the double-stranded molecules (leaves single stranded templates anchored to substrate)
- complete amplification
- determine first base
- image first base (after laser excitation, emited fluorescence from each cluster is captured)
- determine second base
- image second chemistry cycle
- sequencing over multiple chemistry cycles
- align data
Roche 454
sequencing by synthesis: nucleotide incorporation leads to light emission
pyrosequencing advantages
- very fast
- low cost per base
- large throughput; up to 40 megabases/experiment
- no need for bacterial cloning (with its associated artifacts) this is especially helpful in metagenomics
- high accuracy
pyrosequencing disadvantages
- short read lengths (soon to be extended to ~500 bp)
- difficulty sequencing homopolymers accurately
nanopore sequencing
- DNA can be sequenced by threading through a microscopic pore in a membrane
- bases are identifies by the way they affect ions flowing through the pore from one side of the membrane to the other
- electrical current is recorded each type of nt produces different current
nanopore sequencing advantages
- small instrument and portable like a USB flash drive
- long read (a few Kb)
- real time
- low cost
- single molecule sensitivity
nanopore sequencing disadvantages
- low resolution (DNA moves too fast through nanopores)
- feed DNA to nanopore progressively (one solution: integration of the exonuclease and the nanopore detection systems
microarray
- large scale, high throughput
- based on complementary DNA hybridization
- visualization by flourescence
microarray applications
- gene discovery (90% of our genes have yet to be ascribed a function)
-> assigning function to sequence
-> discovery of disease genes and drug targets
-> target validation - genotyping
- microbial ID
- SNP profiling and other genome wide study (mostly replaced by DNA sequecing technology
oligonucleotide microarray
measures gene expression levels (at a transcriptional level) in order to improve diseases diagnosis as well as to create new effective treatment regimens
oligonucleotide clips
- probes are synthesized in situ, using combinatorial chemistry and photolithography
- probe cells are square shaped features on the chip containing millions of copies of a single 25-mer probe. sides are 18-50 microns
affymetrix microarrays
~10^7 olgionucleotides, half perfectly match mRNA (PM), half have one mismatch (MM)
raw gene expression is intensity difference: pM-mM
oligonucleotide advantages
- highly hygienic chips since these chips are synthesized by a photolithography process
- probes design is based entirely on sequencing information, there is no need for the physical intermediates, such as bacterial plasmids or PCR products, which results in a minimum chance to create probes mix-up
oligonucleotide disadvantages
- highly cost to design certain chip
- the need to access to expensive specialised equipment to run the analysis
- short sequence nucleotide probes may decrease the sensitivity of binding in comparing with DNA microarray
