Next generation sequencing Platforms, chemistries, and applications

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

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

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

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

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

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)

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?

Bind to beads & PCR amplify in emulsion (ePCR)

Spot beads onto picotitre plate (flow cell)

GS FLX sequencing chemistry

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

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: 200-400 bp Low biological replication (n = 8 for $10k run) Low flexibility in throughput: must do high throughput

Illumina (formerly Solexa) Polymerase-based sequencing by synthesis

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

Clonal amplification of individual molecules

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

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

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: 20-100 bp Low biological replication (n = 8 for $10k run) Low flexibility in throughput: must do high throughput Run lasts from 1-3 days

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

SOLiD chemistry

Coverage: 20X

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: 25-50 bp Low biological replication (n = 8 for $12k run) Low flexibility in throughput: must do high throughput Run lasts from 3-6 days

Platform comparison

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

Hypothetical experimental

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

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. ? ?

Fu et al. 2008

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!!

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

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

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 30-400bp Wang et al. 2009, Nature Genetics

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

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

Marioni et al. 2008

Marioni et al. 2008

Marioni et al. 2008

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

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