Workshop on Genomics (Notes, Day 2) – Sequencing Technologies

Dr. Konrad Paszkiewicz, University of Exeter

Lecture slides:

From Sanger -> human genome -> 2nd gen -> 3rd gen -> Nanopore (not yet released)

Recommended videos on Sanger sequencing:

Illumina – Least expensive and most commonly used

– short run times
– straight-forward sample prep
– open source software

– short reads
– pooling samples
– sequence clusters on flow cell

Each cycle uses -> 1.) blocking, 2.) enzyme, 3.) fluorophore

* Paired-end sequencing is analyzed based on lane proximities, bridge amplification, each dot represents cluster, lane calling

Each cycle looks at color and position of dots:

Screenshot from 2014-01-14 23:04:28

Oversimplification of adding bases at each cycle: dye-labeled bases are added, then block, then block is washed away, laser excitation, the next cycle starts:

Screenshot from 2014-01-14 23:08:09 Screenshot from 2014-01-14 23:08:38

The resulting image of the flow cell, at each cycle, looks like this:

Screenshot from 2014-01-14 23:09:24 Screenshot from 2014-01-14 23:09:52

Algorithms take into consideration emission wavelengths at each cycle at each spot, and the reads are constructed from these. Each cycle looks at color and position of the dots.

36-150 nucleotides per read, 300 Gb data per run

Quality scores decrease as run progresses, error rate of enzyme increases

2 sets of amplifications – before (library prep, enough sample), during (to get enough signal)

– cluster merging (Krueger et al. 2011)
– inverted repeats and GGC motif

454 Sequencing: longer read lengths, bead-based adapters at end of bead, pyrosequencing, phased out by Roche in 2012

SOliD: abandoned by Life Technologies in favor of Ion Torrent recently

Benchtop Sequencing:

Ion Torrent: no optics, pH sensor detects nucleotide by release of H+ (different for each base), no incorporation or termination, 400 bp reads, 2 hr runtime, various chip types

Ion Proton: 200 bp reads, single end, PGM vs. Proton

Useful benchtop technology review paper, Loman et al. (2012):

3rd gen: single molecule sequencing – Pac Bio only one on market at the moment (RSII)
– Zero Mode wave (ZMW) guide on an SMRT flow cell (looks like a microarray with fixed position
– camera detection
– library prep required
– 100,000-150,000 per run, few hours, $500/run
– 300-500Mb
– distribution of read lengths
– good for detecting epigenetic changes
– error rates, but algorithms to correct,
– look at materials on PacBio github:
– Cicular consensus sequencing can be used

Nanopore = “very small hole”
– measures current changes as polymer passes through hole
– exonuclease to chop DNA
– cyclodextrin to measure charge across membrane

Strand sequencing, MinIon:
– 512 pore chip
– 100 Mb-1 Gb per run
– disposable after 6 hr run
– 4% error rate in trials
– requires library prep – introduces loops for feeding to Nanopore
– more for detection
– error profiles can be handled with different pore types with complimentary error
– cost is high

Illumina still least expensive of all technologies.

***Take-home message: Technology is becoming easier to acquire massive amounts of data. Bottleneck is now always at bioinformatics level.

About Lisa Johnson

PhD candidate at UC Davis in Molecular, Cellular, and Integrative Physiology
This entry was posted in Bioinformatics, Genomics Workshop, Sequencing. Bookmark the permalink.

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