Question

Microbiology Tutorial: Sequencing and Gene Expression Analysis

Answer 1

Note: This is a reference for educational/studying purposes, not a question, please save all comments or questions for the end.

Answer 2

\({\bf{genomics}}\) the application of sequencing methods and bioinformatics into the sequencing and analysis of the genome for scientific research esp. on evolution and the study of disease

\({\bf{Two~General~Approaches}}\)
1. "shotgun" method where the genome is spliced, individual parts are sequenced, and the sequences are "stitched" together to create the full genome
2. hierarchical shotgun sequencing where the genome is first divided into fragments, ordered, and each of the individual fragments is divided into segments that are small enough to sequence

first organism sequenced: bacteriophage phi 174, approx 5,400 nucleotides
for reference: human genome is about 3billion base pairs organized in ~20,000 genes

Answer 3

\({\bf{Illumina}}\) (next-gen sequencing, or NGS)
1. sample prep
2. cluster generation
3. sequencing
4. data analysis

- adapters added to ssDNA fragments, oligose binding sites, motifs, indices
- flow cell contains two types of oligose on a glass slide
- oligose binds to DNA fragments on one end
- polymerase copies the fragment into double stranded DNA, denature the strand to separate, then the original template is washed away
- bridge amplification: fragments bend over and bind to the oligose on the other end, repeating the DNA polymerase duplication method, denatured, washed off, leaving only forward strands
- 3' ends blocked to prevent further synthesis
- sequencing by synthesis: fluorescently tagged nucleotides added, each addition causing the emission of light which is recorded to determine the type of nucleotide added

advantages: can be scaled up, can detect SNPs
disadvantages: can take a lot of time, susceptible to substitutions

Answer 4

\({\bf{Microarrays}}\)

- microarray is composed of thousands of cells (not cells in the living sense, cells as in tiny spaces) each with a small DNA sequence
- two samples of cDNA to be compared must be dyed green/red respectively
- each sample will either hybridize or not hybridize with each cell, giving each cell a red/yellow/green/black color based on which genes hybridize (yellow means both, black means neither)
- unhybridized dna gets washed off

advantages: easy to automate, can be used for multiple genes at once
disadvantages: requires knowledge of the organism's DNA sequence ahead of time, cheaper

Answer 5

\({\bf{RNA-seq}}\) (the methodology is very complex but will give a general outline)

- applying NGS methodology to sequence RNA/mRNA/tRNA/rRNA, etc.
- time-sensitive transcript

advantages: dynamic, can detect single nucleotide variants, isoforms, allele specific expression
disadvantages: expensive

Answer 6

\({\bf{Nanopore}}\)

-feed DNA through nanopore in cell
- DNA is mixed with processive enzymes which guide the DNA through pore
- as nucleotide is processed, the electric force is perturbed which produces a signal which can be detected and analyzed

- advantages: doesn't require polymerase/ligase/etc. can work on longer sequences
- disadvantages: doesn't work on all sequences

Answer 7

\({\bf{Sanger~Sequencing}}\)

- separate cDNA into 4 equivalent groups (1 for each nucleotide)
- each group is given dNTPs, ddNTPs in small concentrations of ~1% (missing 3' OH so no bases can be attached after the ddNTP is attached), DNA polymerase, radioactive primers
- DNA polymerase synthesizes complementary strand, will occasionally pick a ddNTP which stops further transcription, fragment is cleaved
- fragments are analyzed through gel electrophoresis to determine relative nucleotide base content

advantages: cost-efficient esp. for smaller regions, low error rate
disadvantages: can take a long time

Answer 8

\({\bf{Applications~and~Potential~Problems}}\) (mostly a healthcare/privacy thing)

- positives: individualized healthcare profiles based on one's genome, genetic counseling, reduction of heritable diseases passed down
- negatives: privacy issues with data, people being discriminated against for genetic problems, insurance rates going up

Answer 9

This is the end of my tutorial; I hope you found it helpful. If you have any ∗relevant∗ comments or questions I will attempt to address them to the best of my ability.

Thank you for reading!