Applied Genomics Lectures

Ligate vs Hybridize

• Ligate: to adhere to or “stick on”

• Hybridize: to adhere to or attach to (usually at a specific complementary sequence)

Paired End Reads

Sequence of DNA from both ends of a DNA fragment

Long-Read Sequencing

Does not result in any unknown DNA in the read.

contig

Continuous stretch of DNA sequence that has no missing or unknown nucleotides

Scaffold

• Scaffolds join contigs together

• They are able to span unknown sequence

• To do this they either use paired ends, or some other technology to identify the order and orientation of the contigs

Genome Sequencing and Assembly Short Process

• Genome Sequencing: multiple copies of gene, sheared random fragments, size fractionated fragments, reads

• Genome Assembly: contigs, scaffolds

Recap Steps of Genome Sequencing

Summary of Genome Sequencing

• Many copies of the genome are used for sequencing unless “Single Cell Sequencing” is applied

• Unless primers are used sequencing cannot target a particular gene region

• Paired-end sequencing reads a single random DNA fragment from both 5’ to 3’ and 3’ to 5’ directions

• Only the ends of the fragment are read with the shorter read technologies

• Long-read sequencing does not use paired ends

Depth of Coverage

The average number of times each base of a genome is sequenced or read. We want:

• 10-15x for animals

• 200x for humans

Rule-of-Thumb for a Variant to be Confirmed

To believe that a variant is really a variant and not just a sequencing error, we need to see it at least twice

• Grey shows the unknown sequence so overlaps of black help to create a known scaffold

• Contigs are not always assembled in the same orientation

• We expect half will be 5’ to 3’ and half will be 3’ to 5’

• So we may need to flip them over to make them match properly on the scaffold

N’s in Genome Assembly

When the number of base pairs in a gap is known then it is replaced by the same number of ‘N’ - if it is unknown then it is replaced by about 200 N’s.