The Woogie 1

Question 1

Cons of sanger sequencing?

Answer 1

1. expensive
2. error prone
3. prone to bias
4. Low throughput (low amounts of reads can be sequenced at one time)

Question 2

Alignment techniques using reference genome

Answer 2

1. Align reads against reference genome
   2.Mapping: recover position of sequence reads in genome
2. Alignment: recover position of sequence read in genome read
3. Alignment based on sequence matches to locate gene read

Question 3

Genome Project Challenges

Answer 3

1.Sequence technologies not perfect
2. DNA harder to sequence between samples
3. Reference genome could have multiple organisms which means bigger reads.
4. Sample sequence error/mismatches/gaps.

Question 4

2nd Generation Sequencing methods

Answer 4

1. Library prep: Cells -DNA
2. DNA fragments attach to adapters
3. Anchoring to sequencer
   - DNA melted added to flow cell
   - Floor cell saturated with oligonucleotides, complimentary to the adapters.
4. Adapters sequence attach fragments to the bottom of the machine

Question 5

First generation: Sanger sequencing- sequencing by synthesis

Answer 5

1. Fluorescent bases added by polymerase
2. Laser Identifies base type
3. Fluorescent base then removed
   4.Terminal nucleotide can accept another base

Question 6

Genome Size Abundance

Answer 6

1.Complex Organisms generally have larger genomes. Less Complex smaller.

Question 7

What is in a genome?

Answer 7

1.Genes
2.Centromeres and telomeres
3. Bind site, repeat region transposable elements
4. Epigenetics marks:- methyl markers in histones

Question 8

Sanger sequencing using dNTP and ddNTP

Answer 8

1. Unwind DNA helix to single strands
2. Polymerase makes new strand
3. ddNTP halts polymerase action
4. Sequences run on gel (electrophoresis)
5. Laser shines onto ddNtP nucleotides

Question 9

Pro’s of 2nd Gen sequencing

Answer 9

1.Fast
2.Cheap
3.High throughput

Question 10

3rd Generation Sequencing Pros

Answer 10

1. Fast
2. Cheap
3. High throughput
4. Can read single strands
5. Can interpret long reads

Question 11

What is throughput sequencing?

Answer 11

The the computers ability to test a specific number of sequences at one time?

Question 12

Cons of 2nd generaton sequencing

Answer 12

1.Prone to error (repeat regions)
2.Prone to amplification biases
3.Fragment length restricted

Question 13

A Genome consists of?

Answer 13

1.Chromosomes
2. mtDNA
3. Chloroplast DNA
4. Plasmids

Question 14

3rd Generation Sequencing Cons

Answer 14

1. Some sequencing can be expensive
2. Nanopore cheap, but flow cell expensive
3. Prone to error (only 80% accuracy)

Question 15

N50 contig read

Answer 15

Point at which genome is covered by contigs of this size
1. ADD up all contigs lengths
2. Sort by decreasing length
3. largest contig
- covers 50% assembly length
- if not take length of second largest

Question 16

Genome assembly metrics depend on:

Answer 16

1. Number of contigs
2. Length of assembly
3. Number of genes
4. Assembly accuracy

Question 17

String graph assembly

Answer 17

1. uses overlap of reads to build graph.
2. reads becomes nodes and overlap become edges
3. need to specify minimum overlap to have meaningful graph to consider as true.

Question 18

De Bruijn Graph

Answer 18

1. Split reads into 2 strings of letters (kmers)
2. Use Kmer overlap to build graph
3. De Bruijn uses short strings and nodes to build graph
4. Repeat regions can make this complicated

Question 19

Presence of necessary of biological genes

Answer 19

90% of species have 1 copy of genes

Question 20

Challenges in genome sequencing

Answer 20

1. Short reads around 300bps
2. Similar sequence multiples found in genome
3. Repetitive regions increase read length
4. Multiple gene copies, sequence errors and uneven genomic coverage

Question 21

Assembly vs alignment

Answer 21

1. Assembly: without genome assembles reads into a genome
2. Alignment: aligns reads against given genome

Question 22

Genome assembly

Answer 22

1. Reads look to be made into one piece (hard to achieve)
2. Can assembly into contiguous
3. String between contiguous are scaffolds (NNN)
4. Overall, assemblies exist as contiguous, scaffolds and chromosomes

Question 23

Gene Annotation

Answer 23

1.Assemblies useful when genomic features are identified(gene location/promoters)
2. 1st annotation level (start/stop) codons. 2nd use other organism genes to confirm annotation
3. Use RNA sequence from other species to align against