1. Next generation sequencing
Platforms, chemistries, and applications

2. Outline Sanger sequencing “Next generation sequencing” (NGS)
Chain termination with modified dNTPs
“Next generation sequencing” (NGS)
“Sequencing by synthesis” systems
Pyrosequencing refers to Roche GS FLX (formerly “454”)
3rd generation sequencing (discussed by Kristen)
e.g., Nanostring

3. Sanger sequencing Method of choice for years
Based on chain-terminating nucleotides
Automated by Applied Biosystems using fluorescently-labeled chain terminators
Capillary

4. Method Extract DNA Shear/digest and clone
PCR amplify (cloning optional)
Sequencing reaction
primer
DNA polymerase
regular dNTPs
fluorescently-tagged, chain-
terminating dNTPs
Imaging
CCD reads fluorescence as
fragments pass through capillary

5. Sanger sequencing: pros & cons
Long read lengths: up to ~700 bp
Most flexible in throughput: from 1 to 1,000s of samples
Convenient: found in many facilities
Cons
Expensive: ~$3/sequence
Requires PCR or bacterial-mediated pre-amplification
Cannot quantify genome copies or transcripts from DNA/cDNA libraries*
*Unless doing SAGE

6. Next generation sequencing
Definition: massively parallel, cloning-free sequencing (by synthesis)
Roche GS FLX (pyrosequencing)
Illumina (Solexa sequencing)
Applied Biosystems (SOLiD)

7. Roche GS FLX (“454”) The original “pyrosequencer”
Pyrosequencing is not new (Nyren 1996)
Was converted into high-throughput system in 2005 (Margulis et al. Nature)

8. GS FLX library preparation
Shear DNA/cDNA and ligate to adaptors
Amount of shearing is dependent on desired read length
New reagents “claim” reads up to 500 bp
How much variation does this lead to?

9. Bind to beads & PCR amplify in emulsion (ePCR)

10. Spot beads onto picotitre plate (flow cell)

11. GS FLX sequencing chemistry

12. Output Creates an image for every read ~13 Mbp/hr, ~400-500 bp/read
Best instrument for de novo work

13. GS FLX pros & cons vs. Sanger
Cloning-free
Generates Mbp of DNA sequence
Massively parallel: all sequencing done simultaneously
Quantitative: # reads => # molecules in sample
Cheaper than Sanger at $/bp
Cons
Shorter read lengths: bp
Low biological replication (n = 8 for $10k run)
Low flexibility in throughput: must do high throughput

14. Illumina (formerly Solexa)
Polymerase-based sequencing by synthesis

15. Protocol Shear DNA/cDNA and link to adaptors
Adaptors bind to probes on flow cell
Adaptor “lawn” (similar to a probe array)

16. Clonal amplification of individual molecules

17. Sequencing chemistry Fluorescently labeled bases
Initially blocked to prevent polymerization
Laser reads fluorescence
Unblocked so that next base can be added

18. Output
Superimposed image of 4 colors
RNA-seq application (Kristen)

19. Illumina : pros & cons vs. Sanger
Cloning-free
Generates Gbp of DNA sequence
Massively parallel: all sequencing done simultaneously
Quantitative: # reads => # molecules in sample
Cheaper at $/bp
Cons
Short read lengths: bp
Low biological replication (n = 8 for $10k run)
Low flexibility in throughput: must do high throughput
Run lasts from 1-3 days

20. Applied Biosystems SOLiD
Supported oligonucleotide ligation and detection system
Similar to FLX but uses DNA ligase
ePCR beads coated onto slide

21. SOLiD chemistry

22. Coverage: 20X

23. SOLiD : pros & cons vs. Sanger
Cloning-free
Generates Gbp of DNA sequence
Massively parallel: all sequencing done simultaneously
Quantitative: # reads => # molecules in sample
Cheaper at $/bp
Cons
Short read lengths: bp
Low biological replication (n = 8 for $12k run)
Low flexibility in throughput: must do high throughput
Run lasts from 3-6 days

24. Platform comparison

25. Applications Genome sequencing Resequencing
Transcriptome characterization
Comparative transcriptomics
miRNA profiling
Epigenetics
CHiP sequencing

26. Hypothetical experimental

27. Hypothetical experiment
Sequence cDNA libraries from each bucket and/or treatment
Count reads for each transcript
Compare transcript abundances between treatments
BLAST against reference genome

28. NGS vs. microarray
With microarray: must have sequences in hand to design probes.
With NGS: there is no such bias.
Sequence everything.
# of reads is proportional to # of transcripts.
Also no bias to particular gene region. ? ?

29. Fu et al. 2008

30. Microarrays: a dying technology?
Must generate sequences first
Difficulty in interpreting data
Probe hybridization issues
Can only resolve large differences
NGS shows higher correlation w/ protein
But NGS is a bioinformatics nightmare!!

31. The beginning of the end of the microarray?
Knowledge of sequences on array
Cross-hyb problematic if seq are similar
Difficult to detect low abundant species
Reproducibility b/w labs and platforms

32. RNA-Seq: a new tool for transcriptomics
- “shotgun transcriptomic sequencing/short read”
- more precise method of measuring expression
Illumina, Applied Biosystems SOLiD, 454 Life Sciences
Transcriptomics on non-model organisms
Reveal SNPs
Reveal connectivity b/w exons (long or paired reads)
High accuracy, on par with qPCR
Quantitation
Spike-in RNA standards
No upper limit, 5 orders of magnitude
No extensive normalization required across treatments

33. Wang et al. 2009, Nature Genetics
Total RNA or polyA(+) RNA
cDNA production
Adaptor ligation (one or both ends)
Pair-end or single-end reads
Reads bp
Wang et al. 2009, Nature Genetics

34. Illumina sequencing ~35bp, single end reads, ~ 15 M reads
Nagalakshmi et al. 2008, Science

35. RNA-Seq pitfalls Difficulty with the following:
Mapping short reads to the genome
Appropriate assign. of ‘multi-mapping’ reads
Identification of new splice junctions
Sample comparison to ID diff. exp. genes
Reads mapping outside annotated boundaries
Genomic DNA contamination
Pre-spliced heterogeneous nuclear RNA
Bioinformatic challenge
Shendure 2008, Nature Methods

37. Marioni et al. 2008

38. Marioni et al. 2008

39. Marioni et al. 2008

40. NanoString Technology
Minimal background signal
No amplification (induce bias)
Less sample needed
Improved detection of low exp. RNAs
single copy per cell
Fortina and Surrey 2008, Nature Biotechnoloy

41. Probe Design 2 ssDNA probes/ mRNA (35-50 bp oligo)
Overnight hybridization to mRNA (solution-based)
Slide adhesion via biotin labeled capture probe
Reporter probe, 4 spectrally distinct dyes, 7 spaces
‘Barcode’, 47 or 16,384 barcodes