next generation sequencing (ngs)02710/lectures/sequencing15.pdfnext generation sequencing (ngs)...
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Computational Genomics
Next generation sequencing (NGS)
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Nature Methods 2011
Sequencing technology defies Moore’s law
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2009: Illumina,
Helicos
40-50K$
4
2010: 5K$,
a few days
Sequencing the Human Genome
Year
Log
10(p
ric
e)
2010 2005 2000
10
8
6
4
2 2014: 1000$,
<24 hrs
2008: ABI SOLiD
60K$, 2 weeks
2007: 454
1M$, 3 months
2001: Celera
100M$, 3 years
2001: Human Genome Project
2.7G$, 11 years
2014
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Applications of next-generation sequencing
Nature Biotechnology 26 (10): 1135-1145 (2008)
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Roche & illumina analyzers
Genome Sequencer FLX Illumina Genome Analyzer
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High Parallelism is Achieved in
Polony Sequencing
Polony Sanger
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Generation of Polony array:
DNA Beads (454, SOLiD)
DNA Beads are generated using Emulsion PCR
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Generation of Polony array:
DNA Beads (454, SOLiD)
DNA Beads are placed in wells
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Generation of Polony array:
Bridge-PCR (Solexa)
DNA fragments are attached to array and
used as PCR templates
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Sequencing: Fluorescently
labeled Nucleotides (Solexa)
Complementary strand elongation: DNA Polymerase
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Sequencing: Fluorescently
Labeled Nucleotides (ABI SOLiD)
Complementary strand elongation: DNA Ligase
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Single Molecule Sequencing:
HeliScope
• Direct sequencing of DNA molecules: no amplification stage
• DNA fragments are attached to array
• Potential benefits: higher throughput, less errors
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Technology Summary
Read length Sequencing Technology
Throughput (per run)
Cost (1mbp)*
Sanger ~800bp Sanger 400kbp 500$
454 ~400bp Polony 500Mbp 60$
Solexa 75bp Polony 20Gbp 2$
SOLiD 75bp Polony 60Gbp 2$
Helicos 30-35bp Single molecule
25Gbp 1$
*Source: Shendure & Ji, Nat Biotech, 2008
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Errors
Erlich et al. Nature Methods 5: 679-682 (2008)
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Genome Sequencing
• Goal
figuring the order of nucleotides across a genome
• Problem
Current DNA sequencing methods can handle only short stretches of DNA at once (usually between 100-200 bp’s)
• Solution
Sequence and then use computers to assemble the small pieces
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Genome Sequencing
20 20
ACGTGGTAA CGTATACAC TAGGCCATA
GTAATGGCG CACCCTTAG
TGGCGTATA CATA…
ACGTGGTAATGGCGTATACACCCTTAGGCCATA
Short fragments of DNA
AC..GC
TT..TC
CG..CA
AC..GC
TG..GT TC..CC
GA..GC
TG..AC
CT..TG
GT..GC AC..GC AC..GC
AT..AT
TT..CC
AA..GC
Short DNA sequences
ACGTGACCGGTACTGGTAACGTACA
CCTACGTGACCGGTACTGGTAACGT
ACGCCTACGTGACCGGTACTGGTAA
CGTATACACGTGACCGGTACTGGTA
ACGTACACCTACGTGACCGGTACTG
GTAACGTACGCCTACGTGACCGGTA
CTGGTAACGTATACCTCT...
Sequenced genome
Genome
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Assembly
• How do we use the short reads to recover the genome being sequenced?
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1: Using a reference genome: Alignment of reads to a reference
..ACTGGGTCATCGTACGATCGATCGATCGATCGATCGGCTAGCTAGCTA..
..ACTGGGTCATCGTACGATCGATAGATCGATCGATCGCTAGCTAGCTA..
Reference
Sample
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2. Assembly without a reference (de novo assembly)
• Paired-End sequencing (Mate pairs)
– Sequence two ends of a fragment of known size.
– Currently fragment length (insert size) can range from 200 bps – 10,000 bps
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Velvet
• Euler / De Bruijn approach.
• Introduced as a alternative to overlap-layout-consensus approach in capillary sequencing.
• More suited for short read assembly.
• Based on De Bruijn graph.
• Implemented in Velvet1, the mostly used short read assembly method at present.
1Daniel Zerbino and Ewan Birney. Velvet: Algorithms for De Novo Short Read Assembly Using De Bruijn Graphs. Genome Res. 18: 821-829. 2008
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De Bruijn graph method
• Break each read sequence in to overlapping fragments of size k. (k-mers)
• Form De Bruijn graph such that each (k-1)-mer represents a node in the graph.
• Edge exists between node a to b iff there exists a k-mer such that is prefix is a and suffix is b.
• Traverse the graph in unambiguous path to form contigs.
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De Bruijn graph
• K = 4
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De Bruijn graph method / Velvet
• Elegant way of representing the problem.
• Very fast execution.
• Error correction can be handled in the graph.
• De Bruijn graph size can be huge.
– ~200GB for human genomes.
• Does not use pair information in initial phase, resulting in overly complicated graphs
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Applications (beyond DNA)
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CSIRO. INI Meeting July 2010 - Tutorial - Applications
RNASeq
Library
Construction
Sample
Total RNA
PolyA RNA
Small RNA
Sequencing
Base calling & QC
Mapping to
Genome
Assembly to
Contigs
Digital “Counts”
Reads per kilobase per million
(RPKM)
Transcript structure
Secondary structure
Targets or Products
Reference
PUN
Analysis
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RNASeq – Compositional properties
Depth of Sequence
• Sequence count ≈ Transcript Abundance
• Majority of the data can be dominated by a small number of highly abundant transcripts
• Ability to observe transcripts of smaller abundance is dependent upon sequence depth
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Normalization
• Normalization is the process in which components of experiments are made comparable before statistical analysis.
• It is important in sequencing as well as for microarrays.
• A couple issues in normalization are different sequencing depth (library size) and distributions of reads (long right tails).
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Summarized Counts
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Simple RPKM Normalization
• Proportion of reads: number of reads (n) mapping to an exon (gene) divided by the total number of reads (N), n/N.
• RPKM: Reads Per Kilobase of exon (gene) per Million mapped sequence reads, 109n/(NL),
where L is the length of the transcriptional unit in bp (Mortazavi et al., Nat. Meth., 2008).
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RNASeq - Correspondence
• Good correspondence with :
– Expression Arrays
– Tiling Arrays
– qRT-PCR
• Range of up to 5 orders of magnitude
• Better detection of low abundance transcripts
• Greater power to detect
– Transcript sequence polymorphism
– Novel trans-splicing
– Paralogous genes
– Individual cell type expression
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ChIPSeq
MNase
Linker Digest
Sequence &
Align
Remove
Nucleosomes
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ChIP-Seq
Hongkai Ji et al. Nature Biotechnology 26: 1293-1300. 2008
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ChIP-Seq Analysis
Peak Detection
Annotation
Sequence Analysis
Motif Analysis
Visualization
Alignment
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Alignment
• ELAND
• Bowtie
• SOAP
• SeqMap
• …
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Peak detection
• FindPeaks
• CHiPSeq
• BS-Seq
• SISSRs
• QuEST
• MACS
• CisGenome
• …
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One sample analysis
Poisson background model is commonly used to estimate error rate ki ~ Poisson(λ0)
ki
Or people use Monte Carlo simulations Both are based on the assumption that read sampling rate is a constant across the genome.
A simple way is the sliding window method
Hongkai Ji et al. Nature Biotechnology 26: 1293-1300. 2008
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FDR estimation based on Poisson and negative binomial model
Hongkai Ji et al. Nature Biotechnology 26: 1293-1300. 2008
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Two sample analysis Reason: read sample rates at the same genomic locus are correlated across different samples.
Hongkai Ji et al. Nature Biotechnology 26: 1293-1300. 2008
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CisGenome two sample analysis
ni =k1i + k2i k1i | ni ~ Binom(ni , p0)
k1i
k2i
Alignment
Exploration
FDR computation
Peak Detection
Post Processing
Hongkai Ji et al. Nature Biotechnology 26: 1293-1300. 2008
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Epigenome
• Protein-DNA interactions [ChIPSeq] – Nucleosome positioning
– Histone modification
– Transcription factor interactions
• Methylation [MethylSeq]
• Impact of NextGen – Whole genome profiling
– Resolution
• Analytical challenges
Image : ClearScience
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Third generation sequencing
3rd generation
Single Molecule Sequencing Technology (tSMS)
No amplification
10Gb os sequence data per 8 day run
Single Molecule Real Time (SMRT) sequencing technology (PacBio RS)
PacBio RS
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Nanopore sequencing