CSB349H1 Lecture Notes - Lecture 3: Dna Microarray, Shotgun Sequencing, Transcriptome
14 May 2018
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Lecture 3(a): Transcription
Sequencing by Synthesis Approach:
• Key Idea:
o Undergo rounds of amplification of DNA sequence
o Measure the incorporation of the second strand into the DNA strand
• Sequencing is done on a substrate – fragments of DNA are mobilized
on a substrate (slide)
o Sequencing technology works via shotgun sequencing:
§ Long strand of DNA is sliced into small fragments;
known as sequence reads
• Sequence reads are ligated to adaptors on both ends which are small
known DNA sequences
o Sequence reads are amplified on the substrate
Add unlabeled nucleotides and enzymes to initiate
phase bridge amplification
• Amplified sequences will give rise to clusters of identical DNA sequences
that form clumps on the substrate
o Sequencing is completed by adding labeled nucleotides
(each color specific to a base)
§ Second strand synthesis
will occur which will
incorporate bases represented in variable colors
Transcriptomics:
• Transcriptome is the set of all mRNAs present at a specific time or under specific
environmental conditions
• Two main goals for transcriptome analysis:
a) Identify mRNAs present
b) Determine the relative abundance
Methods for Identifying mRNAs:
a) RNA Sequencing:
o Converting all mRNA present into cDNA and then sequencing cDNA
§ This is because there is no technique that allows us to sequence RNA directly
b) Hybridization (DNA Microarray):
o Short oligos re immobilized and labelled cDNA is hybridized
ð Hybridization methods are easier and more cost effective
ð RNA sequencing is more efficient allowing us to identify transcripts that are not known in
advance
Document Summary
Long strand of dna is sliced into small fragments; known as sequence reads: sequence reads are ligated to adaptors on both ends which are small known dna sequences, sequence reads are amplified on the substrate. Second strand synthesis will occur which will incorporate bases represented in variable colors. Transcriptomics: transcriptome is the set of all mrnas present at a specific time or under specific environmental conditions, two main goals for transcriptome analysis, identify mrnas present, determine the relative abundance. Methods for identifying mrnas: rna sequencing, converting all mrna present into cdna and then sequencing cdna. This is because there is no technique that allows us to sequence rna directly: hybridization (dna microarray), short oligos re immobilized and labelled cdna is hybridized. Hybridization methods are easier and more cost effective. Rna sequencing is more efficient allowing us to identify transcripts that are not known in advance.
