High Throughput Sequencing Technologies. J Fass UCD Genome Center Bioinformatics Core Tuesday December 16, 2014

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1 High Throughput Sequencing Technologies J Fass UCD Genome Center Bioinformatics Core Tuesday December 16, 2014

2 Sequencing Explosion

3 Sequencing Explosion 2011 PacBio Oxford Nanopore? adapted from Mardis 2011 Nature 470:198

4 Current Sequencing Technologies Sanger (Roche) 454 Illumina SOLiD PacBio Ion Torrent Complete Genomics now owned by BGI (Illumina) Moleculo = TruSeq Synthetic Long Reads Oxford Nanopore

5 Sanger Sequencing ddntp's (with fluorescent labels) incorporated (along with unlabeled dntp's) in amplification step, resulting in some molecules terminated at every position Gel / capillary electrophoresis orders molecules by length Fluorescent label (color) indicates terminal base identity at each position Read colors, in order, to derive sequence gov/sequencing/education/how/how _10.html ultranet/biologypages/d/dnasequencing.html

6 Current Sequencing Technologies Illumina MiSeq PacBio RS Illumina HiSeq 2000 / 2500

7 Current Sequencing Technologies Oxford Nanopore MinION Oxford Nanopore GridION (on hold?)? Oxford Nanopore Spot-On? RNA, not cdna?) Oxford Nanopore PromethION (bigger MinION?)?

8 Illumina Illumina HiSeq 2000 / 2500 Illumina MiSeq

9 Illumina 8 "Lanes" (two flow cells per HiSeq) (one-lane FC for MiSeq) Surface of flow cell is coated with a lawn of oligo pairs...

10 Illumina Millions of single molecules hybridize to the lawn of adapters dsdna extended by polymerases Cluster Generation: Hybridize Fragments & Extend Adapter Sequence (contains primer)

11 Cluster Generation: Illumina Denature Double-stranded DNA Original strand dsdna is denatured Original template fragment washed away Newly synthesized strand is covalently bound to flow cell New strand discard

12 Illumina Cluster Generation: Covalently-Bound, Randomly Dispersed Single Molecules Resulting covalentlybound DNA fragments are bound to the flow cell surface in a random pattern

13 Illumina Cluster Generation: Bridge Amplification Single-strand flops over to hybridize to adjacent adapter, forming a bridge dsdna synthesized from primer in hybridized adapter

14 Illumina Cluster Generation: Bridge Amplification dsdna bridge now formed each strand covalently bound to different adapter

15 Illumina Cluster Generation: Bridge Amplification dsdna bridge is denatured

16 Illumina Cluster Generation: Bridge Amplification Single strands flop over to hybridize to adjacent adapters, forming bridges dsdna synthesized by polymerases

17 Illumina Cluster Generation: Bridge Amplification Bridge amplification cycles repeated many times

18 Illumina Cluster Generation dsdna bridges denatured Strands in one of the orientations cleaved and washed away

19 Illumina Cluster Generation resulting cluster has ssdna in only one orientation

20 Illumina Cluster Generation Free 3'-ends blocked to prevent unwanted priming

21 Sequencing By Synthesis Illumina Sequencing primer Sequencing primer is hybridized to adapter sequence, starting Sequencing By Synthesis

22 Sequencing By Synthesis Illumina Cycle four FlNTP's + polymerases Image incorporated Fl-NTP's Cleave terminator and dye X (HiSeq), 250 (MiSeq)...

23 Illumina Sequencing By Synthesis

24 Illumina Sequencing By Synthesis

25 Illumina Paired-end sequencing Bridge amplification to generate strands with opposite orientation

26 Illumina Paired-end sequencing dsdna bridges denatured Strands in already sequenced orientation cleaved and washed away

27 Illumina Paired-end sequencing strands with uniform orientation, opposite that in first read

28 Illumina Paired-end sequencing Free 3'-ends blocked to prevent unwanted priming

29 Paired-end sequencing Illumina Sequencing primer Sequencing primer is hybridized to other adapter sequence, starting second read's Sequencing By Synthesis

30 Illumina HiSeq 2000 stats: Dual surface imaging Fast scanning and imaging Two flow cells (in sequence) Initially: 200 Gbp per run Currently: up to 1Tbp per run Run time 7-8 days (100bp PE) 1-2 day rapid mode 25 Gbp / day 2 billion paired-end reads ( million clusters per lane) < $5k per human genome < $100 per transcriptome

31 Illumina

32 PacBio

33 PacBio RS (Real-time Sequencer) Polymerase / DNA complex adhered to bottom of imaging well (Zero Mode Waveguide)... evanescent wave illuminates tiny volume around polymerase.

34 PacBio RS (Real-time Sequencer) Fluorescently-tagged nucleotides are only seen (for an appreciable amount of time) when associated with polymerase. Persistent time in the excitation volume can be recognized as a "pulse."

35 PacBio RS (Real-time Sequencer) Science, 02 January 2009/ /science

36 PacBio SMRTbell Construct

37 PacBio Sequencing

38 PacBio Sequencing

39 PacBio Sequencing polymerase SMRT bell template sequenced strand

40 PacBio Sequencing

41 PacBio detection of modified bases Movie trace pulse timing can reveal nucleotide modification, e.g. N6-methyladenosine

42 PacBio accuracy

43 Current Sequencing Technologies Oxford Nanopore MinION Oxford Nanopore GridION (on hold?)? Oxford Nanopore Spot-On? RNA, not cdna?) Oxford Nanopore PromethION (bigger MinION?)?

44 Oxford Nanopore

45 Oxford Nanopore

46 Oxford Nanopore

47 Oxford Nanopore hairpin dsdna (green & purple strands) semi-conductor layer pore protein 1D read (if stopped here) on its way to becoming a 2D read

48 Oxford Nanopore hairpin dsdna (green & purple strands) Five or six nt s read at once; basecalls depend on HMM (or something like that).

49 Oxford Nanopore Whole genome shotgun sequence data of E coli MG1655 strain published on GigaDB by Nick Loman. Mean read length ~6 Kbp Max read length ~40 Kbp Errors largely indels error rate comparable to PacBio (?) stay tuned!

50 ... Oxford Nanopore? Sequencing Explosion PacBio RS II 2013 adapted from Shokralla 2012 Molecular Ecology 21:1794

51 Tech Comparison Ryan Kim, ~Dec. 2012

52 Tech Comparison Non-technology considerations error modes related to application single-molecule preferred? novel isoforms... software evolving haplotype determination (phasing) base modification local expertise (!) library prep secondary analysis Availability / turnaround time

High Throughput Sequencing Technologies. UCD Genome Center Bioinformatics Core Monday 15 June 2015

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