Lecture 5

Question 1

Describe ways to improve assemblies

Answer 1

Scaffolding

Question 2

What is Scaffolding

Answer 2

Used to figure out how contigs are connected

Question 3

What are the 2 methods of scaffolding

Answer 3

1. Paired-end Illumina sequencing
2. Long Read Sequencing

Question 4

What is Paired-end Illumina Sequencing

Answer 4

Sequence again in opposite direction
If pairs aren’t perfectly complementary → sequencing errors are present and can be removed from data

Question 5

What is Long Read Sequencing

Answer 5

-3rd gen that helps w/ scaffolding
-Can help fill in gaps

Question 6

What is unique about PacBio Single Molecule Real Time Sequencing (SMRT) –> Long Read Sequencing

Answer 6

-Single Molecule → can see 1 single molecule at a time, no amplification is needed (no clusters)
-50,000 bp
-No pausing or reversible terminators to slow down polymerase to take a picture, it does it in real time

Question 7

What is unique about Oxford Nanopore –> Long Read Sequencing

Answer 7

-Single Molecule
-Long reads → 100,000 bp
-Uses very small pore to block ions and identifies base as it passes through the pore

Question 8

What % of human genome are genes and everything else?

Answer 8

-Genes: 1.5%
-Everything else: 98.5%

Question 9

What are the 3 strategies for identifying genes?

Answer 9

1. Inspection (Bioinformatic)
2. Homology (Bioinformatic)
3. Experiment (Wet lab)

Question 10

What is the homology strategy

Answer 10

-Compare with other genomes
-Search databasesin BLAST to see if that sequence is a confirmed protein coding gene in other organisms

Question 11

What is % identity in the homology strategy

Answer 11

the % of positions that have the same base or amino acid

Question 12

What is the experiment strategy

Answer 12

-RNA-seq: extract RNA, convert to DNA, and shotgun sequence it and see which fragments match to transcribed gene
-Genome wide (whole genome) → not all genes are transcribed all the time
Confirms if something is a gene, but not if something is not a gene

Question 13

What is the ORF (open reading frame)

Answer 13

Sequence of codons without stop codons that can encode protein (Sequence in between start and stop codon)

Question 14

Explain the goals of comparative genomics

Answer 14

-Understand relationships between species
-Helps us understand historical questions and how we can predict evolution or how organisms will change in the future

Question 15

Why do we need to mask introns when trying to find ORF in the inspection strategy

Answer 15

Can be problematic since they may have stop codons that do not affect the protein but are never translated
We hide the stop codons in introns when trying to find genes before translation

Question 16

What features do we look for that are associated with transcription that are upstream of the gene → consistent patterns

Answer 16

-Promoters
-CpG Islands → CG pairings, a lot upstream of genes, not common everywhere else

Question 17

How do we find an ORF?

Answer 17

1. Mask introns
2. Find ORF by length, get rid of all the small ones
3. Look for features associated w/ transcription

Question 18

What 2 ways can we mask introns?

Answer 18

1. Codon bias
2. Consensus plot