Class 02: Whole genome sequencing

The seminal papers

www.cs.arizona.edu/people/gene/#papers

• ``Is Whole Genome Sequencing Feasible?''

• ``Whole-Genome DNA Sequencing''

• ``A Whole-Genome Assembly of Drosophila''

Shotgun sequencing

• Multiply target sequence

• Break sequences into random fragments

• Sort by size, discard big and small pieces

• ‘Insert into bacterial virus (‘vector’)

• Infect bacterial, and let it reproduce, ‘cloning’ the insert

• ‘Read’ the insert

Definitions

• G – length of target sequence

• L – avg length of read

• R – number of sequencing reads

• N – base pairs sequences = RL

• I – avg length of clone inset

• c – N/G = avg sequence coverage

• m – RI/2G, avg clone or map coverage

Problems

• Incomplete coverage

• Sequencing errors (< .01, avg)

• Unknown orientation

• Repeated sequences

Repeat problem

• Repeats vary in length, number, fidelity• Length: few bp to thousands• Number: highly variable, even by individual• Fidelity: sometimes 1-2% variation, or less

(multiple copies, pseudogenes)• Long, infrequent, hi-fi repeats are the biggest

problem

Overlap phase

• Compare every read (in both orientations) to every other

• Accept weighted agreement, bounded by fixed epsilon

• Exact solution is tractable

• Result is overlap graph, with each read a node, each overlap an edge

Layout phase

• Determine pairs which position each fragment

• In graph theoretic terms, find a spanning forest

• Optimal spanning forest is NP-hard

• Variation on greedy is commonly used

Consensus phase

• Problem: find consensus of multiple alignment of reads

• Initially, use overlaps in the spanning forest

• Apply one of several algorithms to refine this

Mates & contigs

‘Double-barreled’ shotgun

• Choose inserts of length at least two ‘reads’

• Sequence both ends (we know their relative orientation and distance)

• Used to order and orient contigs

• Use a supplementary process to fill in the gaps between contigs

Clone by clone (HGP)

Whole genome assembly

• Mates can resolve short repeats

• Problem when you ‘exit’ the repeat: you don’t know which is right

• Resolve using a mate pair which has a read in the unique flanking sequence

Whole genome (illustr)