Next Generation Sequencing. Josef K Vogt Slides by: Simon Rasmussen

Transcription

1 Next eneration Sequencing Josef K Vogt Slides by: Simon Rasmussen 2017

2 Second generation sequencing 454 Illumina % market share SOLiD Ion Torrent (PM)

3 Library preparation 1.reate library molecules 2.Amplification (PR) 3.Massive parallel sequencing DNA from extract Library ragment & polish DNA Adapters Library molecule

4 Amplification and immobilization Emulsion PR (454, Solid, IonTorrent): Water, oil, beads, one DNA template/droplet Bridge PR (Illumina): One DNA template/cluster, primers on surface, grow by bridging primers Bridge PR Metzker, Naten Rev. 2010

5 luorescence detection REVIEWS Illumina - yclic reversible termination Pyrosequencing a Illumina/Solexa Reversible terminators A T A T c Helicos BioSciences Reversible terminators Add all dntps Incorporate all four nucleotides, each label with a different dye labelled w. diff dye T A A T Incorporate single, dye-labelled nucleotides Load template beads into wells reate fourcolor image Wash, fourcolour imaging T A T Wash, onecolour imaging low one dntp across wells Polymerase incorporates nucleotide leave dye and repeat next cycle leave dye and terminating groups, wash T A T leave dye and inhibiting groups, cap, wash Release of PPi leads to light b Repeat cycles d Imaging, next Repeat cycles dntp Metzker, Naten Rev T A

6 groups, wash groups, cap, wash 2: Imaging handout Repeat cycles Repeat b d Illumina 1: T A Illumina 2: T A T A Top: ATT Bottom: Top: Bottom: TAT ATA One-base-encoded probe An oligonucleotide sequence in which one interrogation base is associated with a particular igure 2 our-colour and one-colour cyclic reversible termination methods. a The four-colour cyclic termination (RT) method uses Illumina/Solexa s 3 -O-azidomethyl reversible terminator chemistry 23,101 (B solid-phase-amplified template clusters (I. 1b, shown as single templates for illustrative purposes). ollo imaging, a cleavage step removes the fluorescent dyes and regenerates the 3 -OH group using the reduci tris(2-carboxyethyl)phosphine (TEP) 23. b The four-colour images highlight the sequencing data from tw amplified templates. c Unlike Illumina/Solexa s terminators, 454: the Helicos Virtual Terminators 33 are labelled same dye and dispensed individually in a predetermined order, analogous to a single-nucleotide addition ollowing total internal reflection fluorescence imaging, a cleavage step removes the fluorescent dye and groups using TEP to permit the addition of the next y5-2 -deoxyribonucleoside triphosphate (dntp) an free sulphhydryl groups are then capped with iodoacetamide before the next nucleotide addition 33 (step d The one-colour images highlight the sequencing data from two single-molecule templates. Metzker, Naten Rev. 2010

7 groups, wash groups, cap, wash 2: Imaging handout - answers Repeat cycles Repeat b d T A Quality of base call deteriorates after many T A cycles T A Top: ATT Bottom: Top: Bottom: TAT ATA One-base-encoded probe An oligonucleotide sequence in which one interrogation base is associated with a particular igure 2 our-colour and one-colour cyclic reversible termination methods. a The four-colour cyclic termination (RT) method uses Illumina/Solexa s 3 -O-azidomethyl reversible terminator chemistry 23,101 (B solid-phase-amplified template clusters (I. 1b, shown as single templates for illustrative purposes). ollo imaging, a cleavage step removes the fluorescent dyes and regenerates the 3 -OH group using the reduci tris(2-carboxyethyl)phosphine (TEP) 23. b The four-colour images highlight the sequencing data from tw Homopolymer runs are amplified templates. c Unlike Illumina/Solexa s terminators, the Helicos Virtual Terminators 33 labelled same dye and dispensed individually in a predetermined problematic, order, analogous to a gives single-nucleotide rise to addition ollowing total internal reflection fluorescence imaging, a cleavage step removes the fluorescent dye and groups using TEP to permit the addition of the next y5-2 -deoxyribonucleoside indels triphosphate (dntp) an free sulphhydryl groups are then capped with iodoacetamide before the next nucleotide addition 33 (step d The one-colour images highlight the sequencing data from two single-molecule templates. Metzker, Naten Rev. 2010

8 Illumina: Quality deterioration an you think of why? Efficiency of incorporation: Polymerase incorporation of base Enzyme that cleaves the dye

9 NextSeq/HiSeq3000/4000 hemistry is not based 4 dyes (as before) but 2 dyes T (red), (green), A (both) and (none = dark ) aster processing rate and cheaper reagents Slightly increases error rate Problem with stretches because is not dyed

10 Similar principle to 454 Ion Torrent Library: Emulsion PR Based on semiconductors Ion Torrent Detection is based on H ions (ph) changes

11 Solid

12 Solid example Double-base encoding olorspace Low error rate Errors solid0420_ _ra/1 T !=:369A?:.<9=.-5=%3-:6%3&<2%(169%,0.3%&'&(&.'%%%&&, AAT...

13 omplete enomics ssdna -> DNA nanoballs Use silicon chips with sticky spots Place DNBs into each spot Sequence using ligase and flourescent labeled probes You cant buy the machine - Acquired by BI - delayed - Only humans!

14 3rd generation No amplification (PR introduces bias!) Simple sample preparation Helicos Pacific Biosciences Oxford Nanopore

15 Pacific Biosciences Slowed down DNA polymerase, measure light emission, Long reads > 10kb, high error rate (but random) Pacific Biosciences

16 Oxford Nanopore Nano-scale pores, with current across Drag DNA stretch through the pore Measure change in current (pentamers) Long reads (up to 200k), currently ~5k, some systematic errors Oxford Nanopore

17 Mean Signal (pa) PacBio o tp t A camera records the changing colours from all ZMWs; each colour change corresponds to one base O T o tp t (s iggles) ach current shi as DNA translocates through the pore corresponds to a particular k-mer Synthetic Long Reads (2nd gen) Time (seconds) B S nthetic long-read se encing Ba ll mina Bb Illumina Synthetic long-read sequencing (Moleculo) D ragment DNA is fragmented and selected to ~10 kb n matic clea age DNA is barcoded and fragmented to ~350 bp ~3,000 molecules per well Based on Illumina sequencing 10X enomics A1 A2 enomics m lsion P R Arbitrarily long DNA is mixed with beads loaded with barcoded primers, enzyme and dntps EMs Each micelle has 1 barcode out of 750,000 mplification Long fragments are amplified such that the product is a barcoded fragment ~350 bp Pooling The emulsion is broken and DNA is pooled, then it undergoes a standard library preparation Barcodes DNA from the same well shares the same barcode Using barcode system to create linked reads / read clouds Pooling DNA from each well is pooled and undergoes a standard library preparation Se encing DNA is sequenced on a standard short-read sequencer Lin ed reads All reads from the same EM derive from the long fragment, thus they are linked Reads are dispersed across the long fragment and no EM achieves full coverage of a fragment Stacking of linked reads from the same loci achieves continuous coverage at re Re iews enetics 344 JUNE 2016 VOLUME 17

18 Machine overview - I ompany/technology urrent machine, key characterstics 454 S LX+, 7-800bp length, 1M reads Illumina Solid (Life) PM (Life) omplete enomics (BISEQ) PacBio Oxford Nanopore Illumina synthetic 10X enomics HiSeq400/HiSeqX Ten, bp length, up to 2-4B reads 5500XL, 50/75bp length, 1.5B reads Ion Proton, 200/400bp length, 80M reads BISEQ-500, bp, 200b in total Sequel, 8-12kb length, 350k reads ridion, up to 200kb, >100k reads up to 100kb synthetic length, 1000$ pr. b up to 100kb synthetic length, +500$ per sample Excellent overview at oodwin et al., Nature Reviews (2016)

19 Machine overview - II Benchtop machines ompany/technology urrent machine, key characterstics 454 S Junior, 4-500bp length, 100k reads Illumina MiSeq, 300bp length, 50M reads PM (Life) Ion Torrent, 400bp length, 5M reads Oxford Nanopore MinION, up to 200kb, 100k reads Excellent overview at oodwin et al., Nature Reviews (2016)

20 More NS material Elaine Mardis on NS technology Youtube has many more Excellent review on NS technologies: oodwin et al., Nature Reviews (2016) On ampusnet together with many other papers!