genome sequencing

key steps in a whole genome sequencing project

isolate, fragment, prepare genomic library using adapters, sequence, align and quality filter, map

Illumina read length

short - 250bp

Ion Torrent read lengths

short - 450bp

PacBio read lengths

long - 30kbp

Oxford nanopore read length

long - 30kbp

Illumina run time in hours for 100 nucleotide paired end run, bacterial genome

240 hours

Ion torrent run time (200 nt run, bacterial genome

3 hours

run time PacBio, bacterial genome

3 hours

Which sequencing technologies also have a bench top equivalent?

Illumina and ion torrent

Illumina step 1 - library preparation

Fragment DNA. Adapters ligated onto fragments to immobilise DNA on flow cell

Illumina step 2 - cluster generation

Flow cell coated in complementary adapters. DNA library diluted to distribute fragments and bridge PCR amplifies DNA to form clusters

bridge amplification PCR

• ssDNA binds flow cell adapters in random pattern at one end

• free ends attach surface via primers - ssDNA bends

• polymerase and nucleotides added → reverse strand synthesised

• reverse cut from forward (template remains), one adapter per strand denatures from flow cell

illumina step 3 - sequencing by synthesis

Clusters big enough to view microscopically. Cluster colour indicates base incorporated

alt library prep method

use paired end reads to improve genome assembly. Once sequencer has undergone one round, process repeated from other end

indexing/barcoding

• maximises outputs/spend

• allows multiple samples to be mixed

• reads computationally separated

ion torrent

post light sequencing by proton. Sequential flood of dNTPs released, can see which base is present based on whether hydrogen and pyrophosphate released by polymerase

difference between PacBio and other sequencing technologies

most methods immobilise template DNA whereas PacBio immobilises polymerase at bottom of detector zone

SMRT PacBio process

Adaptor binds polymerase. Fluorescently labelled bases held in detection zone. Different light pulse produced based on what base is added in

oxford nanopore technology

Ionic current passed through protein nanopores. DNA causes disruption in current which can be analysed

PHRED score

automates base calling in Sanger projects. For each increase of 10 in PHRED score base call accuracy increases 1.1x, starting with accuracy 90% at Phred score 10

How does quality change towards the end of a read?

most methods show quality decreae for bases towards end

fast Q line 1 to 4

1 = sequencing platform and conditions
2 = sequence
3 = spacer
4 = PHRED score representation

whole genome sequencing and cancer

since 2021, paired whole genome sequencing carried out by NHS England on cancer patients under 25

WGS outside of DNA sequence

can be used to study RNA and epigenetics