1. Current Sequencing Technologies and Data Generation
Corbin Jones & Piotr Mieczkowski
Department of Biology, College of Arts and Sciences, Carolina Center for Genome Sciences Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill

2. Library prep
Sequencing

3. Pooling for multiplexing
Sample Submission
Data flow
3 µl of Sample
Sonication QC
LIMS
Sample flow
Transfer to new plate/tube
Sample failed y/n
Concentration
Size
Leftover sample
Dilution
Sample flow
Same plate
End Repair
Adenylation
Adapter Ligation
Leftover sample
Size Selection
3 µl of Sample
SAMPLE
PCR QC
15 nM dilution
Facility
Pooling for multiplexing

4. Manual and Semiautomation in HTSF library prep workflow
Sonication
Automated library size selection
Magnetic beads DNA size selection
Sage – Pippin – Automated size selection system

5. Automation in HTSF Tecan – Freedom Evo system – 8 tip
2x48 (96) samples per week – DNA library prep
Automated sample normalization steps
PCR and qPCR preparation
Reagans distribution
Can be adapted to small and medium scale protocols for Illumina and Ion Torrent
We have all necessary components for DNA/RNA extraction using Qiagen kits
Caliper – Sciclone system
96 tip pipetting head
(8-96 samples per run)
TruSeq DNA library preparation
TruSeq Exome Enrichment
SureSelect Agilent DNA capture and library preparation

6. Next-generation Sequencing (Deep Sequencing) Platforms
Short reads
Genome Analyzer IIx (GAIIx), HiSeq2000, HiSeq2500, MiSeq – Illumina
SOLiD 5500xl System – Applied Biosystem
HeliScope™ Single Molecule Sequencer - Helicos
Long reads
Genome Sequencer FLX System (454) – Roche
PacBio RS - Pacific Bioscience
Personal Genome Machine, Ion Proton - Ion Torrent
GridION – Oxford Nanopore
Mapping sequences to large DNA fragments
NABsys
Bionanomatrix

7. UNC – HTSF 9 HiSeq 2000/2500 1 GA II PacBio Ion Torrent
MiSeq (Jeff Dangl)
PIOTR
Liz Buda and Donghui Tan
Also on campus:
454 (Microbiome)
454 jr. (Viral genomics)
MiSeq – Kevin Weeks

8. HiSeq 2000/2500 – 100-160mln single end sequencing reads per lane.
What type of sequencing should I choose for the Illumina sequencing project?
HiSeq 2000/2500 – mln single end sequencing reads per lane.
- ChIPseq – Single End 50 cycles (2-3 human samples per lane)
- RNAseq – Single End 50 cycles (2-3 human samples per lane)
If you are interested in splicing variants and fusion genes both Single End 100cycles and Paired End 2x50cycles will be better option for you.
Whole Genome Sequencing – Paired End 2x100cycles (2-3 lanes per genome)
Exome Capture - Paired End 2x100cycles (4 samples per lane)
MiSeq – 3-7 mln single end sequencing reads per lane. Custom projects , fast turnaround.
Metagenomics - 16S profile – Paired End 2x150cycles up to 24 samples per lane.
Whole Microbial Genome Sequencing - Paired End 2x150cycles

9. SHORT READ PLATFORMS at UNC
HiSeq 2000
Initially capable of up to 600Gb per run in 13 days.
Cost of resequencing one human genome:
Now UNC PI - (30x coverage) about $6,000
Now for outside of UNC - (30x coverage) about $9,000
HiSeq 2500
Initially capable of up 100Gb per run in 27hours.
Cost per genome - ???

10. MiSeq Small capacity system. PE 2x150cycles in 27hours.
PE 2 x 250bp coming soon – error rate for read 1 – less than 1%; read 2 about 1.2%.
In preparation – PE 2 x 400bp – error rate for read1 about 2%; read 2 about 4%.
In preparation – Longer insert size possible 1.5kb

11. PacBio RS Single molecule resolution in real time
Short waiting time for result and simple workflow
Generate basecalls in <1 day
Polymerase speed ≥1 base per second
No amplification required
Bias not introduced
More uniform coverage
Direct observation
Distinguish heterogeneous samples
Simultaneous kinetic measurements
Long reads
Identify repeats and structural variants
Less coverage required
Information content
One assay, multiple applications
Genetic variation (SVs to SNPs)
Methylation
Enzymology
C2 chemistry – installed March 2012
Long reads 6-10kb
Meidan size of molecules 3kb
Still 15% error rate
No strobe sequencing
Software focus on:
De novo assembly
Hi quality CCS consensus reads
In preparation
Load long molecules by magnetic beads
Modified nucleotides detection

12. LS – long sequencing reads CCS – high quality sequencing reads
PacBio RS – two sequencing modes
LS – long sequencing reads
Sample Preparation
Standard
Large insert sizes (2kb-10kb)
Generates one pass on each molecule sequenced
CCS – high quality sequencing reads
Circular Consensus
Small insert sizes 500bp
Generates multiple passes on each molecule sequenced

13. Example Data: 1 smart cell
Pre-Filter # of Bases 180,320,136 bp Post-Filter # of Bases 165,424,592 bp
Pre-Filter # of Reads Post-Filter # of Reads 52801
Pre-Filter Mean Readlength 2399 bp Post-Filter Mean Readlength 3133 bp
Pre-Filter Mean Read Quality Post-Filter Mean Read Quality 0.827
% Adapter Dimer (0-10bp) 1.94 %
% Short Insert (11-100bp) 0.47 %

14. Personal Genome Machine – Ion Torrent (life technologies)
Three types of semiconductor chips:
314 – 20Mb Mb
318 – 1Gb
Read length depends on base composition bp (200cycles)
System is enabled for Paired End 2x100cycles
The fastest sequencing system on the market.
How it works:
H+ ion is released during base incorporation. Individual polymerases attached to beads are positioned in tiny wells that rest on a tiny pH meter.
Recommendation:
Resequencing applications which require fast turnaround of samples
- Amplicons (PCR products)
Small and medium size genomes
Custom DNA capture applications

15. PGM/Ion Torrent Data 316 chip
Thr.
note the homopolymer problems
Total Number of Bases [Mbp] 77.65
‣ Number of Q17 Bases [Mbp] 36.11
‣ Number of Q20 Bases [Mbp] 27.33
Total Number of Reads 368,860
Mean Length [bp] 211
Longest Read [bp] 380

16. Library Preparation from Low Quantities of DNA or RNA
Microfluidics stationary and portable systems
Mondrian SP System – NuGEN Technologies
Human libraries from 5ng of total DNA. Only 10-15% of duplicate reads.
Ultralow DNA library systems
Soon:
Ultralow RNA library systems
Libraries from total RNA with rRNA depletion.
Advanced Liquid Logic from RTP

17. Emerging Sequencing Technologies
Semiconductor sequencing chip
Nanopore / Nanochannel sequencing

18. Ion Proton System Human genome in one day
Cost of reagents $1000 per run
Error rate around 1.2%
Human Genome, RNAseq, ChIPseq
Ion Proton Chip I – 10Gb
(Whole Exome capture experiments)
Ion Proton Chip II – 100Gb
Whole human Genome resequencing

19. Oxford Nanopore – new view on sequencing
Hemolysin – pore - inner diameter of 1nm, about 100,000 times smaller than that of a human hair.

20. Oxford Nanopore DNA sequencing
Error rate 4%, prediction for end of the year 0.1 – 2%.

21. Nanopore array

22. Oxford Nanopore – new concepts
MinION
- 150Mb per run
- Tested 48kb read length
$900 per instrument
500 pores per device
GridION
- XXXMb per run
- Tested 48kb read length
$XXX per instrument
2000 pores per device, soon 8000 pores
Cost per human genome $1500.

23. Oxford Nanopore – applications
DNA sequencing
Protein detection
Protein DNA interaction
Small molecule detection
96 well plates for 96 samples
Controlled time of sequencing

24. Intelligent BioSystems Mini20 System (manufactured by Azco Biotech)
Amplification by rolony method
Sequencing by Synthesis with announced 100 base reads, but expect to compete with Sanger down the road
Designed for clinical labs
20 independent flow cells, no queue for loading, run asynchronously
20M reads/flow cell, 4 GB/ flow cell
Potential problems with repeats
System cost $120K, $150 flow cell (disposable), full costs per sample not clear yet.
Entering early access now, expect commercial shipping late 2012

25. Genia Technologies
Very early stage announcement – Backed by Life Technologies (at least 1 year away)
Describe system as a cross between Ion Torrent and Oxford Nanopore
Electronic “Active Control” technology enables highly efficient nanopore-membrane assembly and control of DNA movement through the channel
Initially used α-Hemolysin and claimed 98% raw accuracy with that but now are using an undisclosed pore for further development.
Claim sensitivity 1-2 orders of magnitude greater than Oxford Nanopore.
Ramping up pore density to 100K pores/chip by end of 2012.
Plan to market a mobile reader for <$1K and per sample costs <$100
Plan early access in late 2012, commercial shipment 2013

26. “caveat emptor!”