This document summarizes several next-generation sequencing platforms and applications. It describes the workflows and chemistries of 454, Illumina, SOLiD, and Ion Torrent platforms. These platforms have significantly reduced the cost of sequencing compared to Sanger sequencing. Common applications include whole genome sequencing, RNA sequencing, sequence capture, and amplicon sequencing. Library preparation requires fragmentation of DNA or RNA, addition of adapters, and amplification prior to sequencing.

1. Next Gen Sequencing Platforms and
Applications
Matthew Tinning
Australian Genome Research Facility
1 August 2012

2. Next-Gen Sequencing
Technologies
Roche GS-FLX Life Technologies SOLiD
Illumina HiSeq Life Technologies Ion Torrent

3. Roche GS-FLX

4. Workflow
Sample Fragmentation
Library Preparation
emPCR Setup
emPCR Amplification
Pyrosequencing
Data Analysis

5. Pyrosequencing

6. emPCR
Emulsion PCR is a method of clonal amplification which allows
for millions of unique PCRs to be performed at once through
the generation of micro-reactors.

7. emPCR
The Water-in-Oil-Emulsion

8. Massively Parallel Sequencing

9. Data Analysis
T Base A Base C Base G Base
Flow Flow Flow Flow
Raw Image Files
Image Base- Quality
Processing calling Filtering
SFF File

10. 454 Platform Updates
GS20 • 100bp reads, ~20Mbp / run
GS-FLX • 250bp reads ~100 Mbp / run (7.5 hrs)
GS-FLX Titanium • 400bp reads ~400 Mbp / run (10 hrs)
GS-FLX Titanium Plus • 700 bp reads ~700 Mbp/run (18 hrs)
GS Junior • 400 bp reads ~ 35Mbp/run (10 hrs)

11. 454 Sequencing Output
• *.sff (standard flowgram format)
• *.fna (fasta)
• *.qual (Phred quality scores)

12. Illumina HiSeq

13. Illumina Sequencing Technology
Robust Reversible Terminator Chemistry Foundation
3’ 5’
DNA
(0.1-1.0 ug) A G
T
C G
A
C
T T
A
C C
G
G A
T
A A
C
T C
C
C G G
A
T
T C
Sample G
A
preparation Cluster growth T
5’
Sequencing
1 2 3 4 5 6 7 8 9
T G C T A C G A T …
Base calling
Image acquisition

14. Platform Updates
Solexa 1G • 18bp reads, ~1Gbp / run
Illumina GA • 36bp reads ~3Gbp / run
Illumina GAII • 75bp paired reads ~10Gbp / run (8 days)
Illumina GAIIx • 75bp paired reads ~40Gbp / run (8 days)
Illumina HiSeq 2000 • 100 bp paired reads ~200 Gbp/ run (10 days)
Illumina HiSeq, v3 SBS • 100bp paired reads ~600Gbp / run (12 days)
MiSeq • 150 paired reads ~1.5 Gb/run (27 hrs)
Maximum yield / day 50,Gbp
~16x the human genome

15. Illumina Sequencing Output
• *.fastq (sequence and corresponding quality
score encoded with an ASCII character, phred-
like quality score + 33)

16. Illumina fastq
1 2 3 4 5 67 8
@HWI-ST226:253:D14WFACXX:2:1101:2743:29814 1:N:0:ATCACG
TGCGGAAGGATCATTGTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTT
GAAAAAAAAAAAAAAAAAATTA
+
B@CFFFFFHHFFHJIIGHIHIJJIJIIJJGDCHIIIJJJJJJJGJGIHHEH@)=F@EIGHHEHFFFFDCBBD:@CC@C
:<CDDDD50559<B########
1. unique instrument ID and run ID
2. Flow cell ID and lane
3. tile number within the flow cell lane
4. 'x'-coordinate of the cluster within the tile
5. 'y'-coordinate of the cluster within the tile
6. the member of a pair, /1 or /2 (paired-end or mate-pair reads only)
7. N if the read passes filter, Y if read fails filter otherwise
8. Index sequence

17. Applied Biosystems SOLiD

18. Sequencing by Ligation

19. Base Interrogations

20. 2 Base encoding
AT

21. emPCR and Enrichment
3’ Modification allows covalent bonding to the slide surface

22. Platform Updates
• 50bp Paired reads ~50Gbp / run
SOLiD 3 (12 days)
• 50bp Paired reads ~100Gbp / run
SOLiD 4 (12 days)
• 75bp Paired reads ~300Gbp / run
5500xl (14 days)
Maximum yield / day 21,000,000,000bp
7x the human genome
3.5 hours of sequencing for a 1 fold coverage.....

23. SOLiD Colour Space Reads
• *.csfasta (colour space fasta)
• *.qual (Phred quality scores)
>853_17_1660_F3
T32111011201320102312......
AA CC GG TT 0 Blue
AC CA GT TG 1 Green
AG CT GA TC 2 Yellow
AT CG GC TA 3 Red

24. Applied Biosystems:
Ion Torrent PGM

25. Ion Torrent
• Ion Semiconductor Sequencing
• Detection of hydrogen ions during
the polymerization DNA
• Sequencing occurs in microwells
with ion sensors
• No modified nucleotides
• No optics

26. Ion Torrent
dNTP • DNA Ions  Sequence
– Nucleotides flow sequentially over Ion
semiconductor chip
H+ – One sensor per well per sequencing
reaction
∆ pH – Direct detection of natural DNA extension
– Millions of sequencing reactions per chip
∆Q – Fast cycle time, real time detection
Sensing Layer
Sensor Plate
∆V
Bulk Drain Source To column
receiver
Silicon Substrate

27. Ion Torrent: System Updates
314 Chip • 100bp reads ~10 Mb/run (1.5 hrs)
• 100 bp reads ~100 Mbp / run (2 hrs)
316 Chip • 200 bp reads ~200 Mbp/run (3 hrs)
318 Chip • 200 bp reads ~1 Gbp / run (4.5 hrs)

28. Ion Torrent Reads
• *.sff (standard flowgram format)
• *.fastq (sequence and corresponding quality
score encoded with an ASCII character, phred-
like quality score + 33)

29. Summary of NGS Platforms
• Clonal amplification of sequencing template
– emPCR (454, SOLiD and Ion Torrent)
– Bridge amplification (Illumina)
• Sequencing by Synthesis
– 454 Pyrosequencing
– Illumina Reversible Terminator Chemistry
– Ion Torrent Ion Semiconductor Sequencing
• Sequencing by ligation
– SOLiD – 2 base encoding
• Dramatic reduction in cost of sequencing
– GS-FLX provides > 100x decrease in costs compared to
Sanger Sequencing
– HiSeq and SOLiD > 100x decrease in costs over GS-FLX

30. Applications
• DNA
• Whole Genome
– Shotgun & Mate Pair
• Sequence Capture
• Amplicon
• RNA
• mRNA
• small RNA

31. Next Gen Sequencing Library
Preparation

32. Sample preparation
mRNA DNA
chemical
mechanical
Fragmentation
cDNA Synthesis Fragmentation
Ligation of Amplification/
Sequencing Adaptors
Library Fragment Size Selection

33. Shotgun Libraries
• Illumina
– Input: 1 ug of DNA
– Fragmentation w/ Covaris
– Size Selection w/ gel excission
• Insert Size 300-400 bp
• gel free method for captures
– PCR “enrichment” (10 cycles)
• 454
– Input 500 ng of DNA
– Fragmentation w/ Nebulization
– Small fragment removal (AMpure
size exclusion)
• Library size ~900 bp

34. Mate-Pair Libraries
• Mate pair libraries for scafolding and
structural variation
– Input: 5-20 ug of DNA
– 3kb, 8kb and 20Kb inserts
– Size Select via gel electrophoresis
– Adaptors for circularization via Cre
recombinase (454)
– PCR amplification (20 cycles)

35. Sequence Capture
• Enrichment for specific targets via
capture with oligonculeotide baits
– Exome Capture
• TruSeq Exome 62 Mb
• NimbleGen SeqCap EZ Exome
Library v2 & v4
• Agilent SureSelect XT/2 All Exon
v4 (+UTRS)
– Custom Capture
• TruSeq Custom Enrichment (700
Kb- 15 Mb)
• NimbleGen SeqCap EZ Choice (up
to 50 Mb)
• Agilent SureSelect XT/2 Custom
(up to 34 Mb)

36. RNA-seq (cDNA libraries)
• Shotgun library of cDNA
– Isolation of Poly(A) RNA
– (100 ng – 4 ug of total RNA)
– Chemical Fragmentation of RNA
– Random primed cDNA Synthesis &
2nd strand Synthesis
– Follows standard “DNA” library
protocol

37. Illumina small RNA
• Illumina Small RNA Sample
Preparation
– Input: 1-10 ug of total RNA
• 50-200 ng of small RNA
– RNA-adaptor ligation before cDNA
synthesis
– Small RNA size selection via PAGE
• Library fragment ~145-160bp
(insert 20-33 nucleotides)
– PCR “amplification” (11 cycles)

38. Sample requirements
DNA – OD260/280 1.8-2.0 RNA – RIN > 8.0
gDNA 1 µg (Illumina)
500 ng (454)
5-20 ug (454 Paired-End)
Total RNA 100 ng- 4 µg (mRNA-seq)
1-10 ug (small RNA)
mRNA 10-100 ng (Illumina)
200 ng (454)
small RNA 50-200 ng