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High Throughput Sequencing the Multi-Tool of Life Sciences
Lutz Froenicke
DNA Technologies and Expression Analysis Cores
UCD Genome Center
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DNA Technologies & Expression Analysis Cores
• HT Sequencing (Illumina & PacBio)
• Illumina microarray (expression analysis, genotyping)
• consultations
• introducing new technologies to the campus
• shared equipment
• teaching (workshops)
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Hi-C
Lieberman-Aiden 2010
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Hi-C
Lieberman-Aiden 2010
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Dovetail Sequencing (Putnam 2015)
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METHYL-Seq
• Bisulfite Conversion
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ChIP-Seq • Chromatin Immuno Preciptation
DNA/Protein interactions e.g. transcription factors
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DRIP-Seq
• R-loops: three-stranded structure Genome-wide mapping of RNA-DNA hybrids
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Complementary Approaches
Illumina PacBio
Still-imaging of clusters (~1000 clonal molecules)
Movie recordings fluorescence of single molecules
Short reads - 2x300 bp Miseq Up to 60 kb, N50 21 kb
Repeats are mostly not analyzable spans retro elements
High output - up to 100 Gb per lane up to 1,3 Gb per SMRT-cell
High accuracy ( < 0.5 %) Error rate 15 %
Considerable base composition bias No base composition bias
Very affordable Costs 5 to 10 times higher
De novo assemblies of thousands of scaffolds
“Near perfect” genome assemblies
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RNA-seq Gene Expression
Small RNA
ChIP-SEQ
Genotyping
De novo genome Sequencing
DNA Methylation
Metagenomics
Exome Sequencing
Splice Isoform Abundance
Genome Resequencing
3D Organization
SNPs, Indels CNVs
Rearrangements
High
Throughput
Short Read
Sequencing:
Illumina
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Rearrangements
De novo genome Sequencing
RNA-seq Gene Expression
Small RNA
ChIP-SEQ
Genotyping
DNA Methylation
Metagenomics
Exome Sequencing
Splice Isoform Abundance
Genome Resequencing
3D Organization
Long Read
Sequencing
PacBio
SNPs, Indels CNVs
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Illumina sequencing workflow
Library Construction
Cluster Formation
Sequencing
Data Analysis
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DNA, RNA
RNA
Fragmentation
Mechanical shearing:
• BioRuptor • Covaris
Enzymatic:
• Fragmentase, RNAse3
Chemical: Mg2+, Zn2+
DNA, RNA
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DNA library construction
5
’
5
’
5
’
5
’
5
’ 5
’
HO P
P
OH
5
’
5
’ A
P P
A
T T
Fragmented DNA
End Repair
Blunt End Fragments
“A” Tailing
Single Overhang Fragments
Adapter Ligation
DNA Fragments
with Adapter Ends
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Enrichment of library fragments
5’
5’
PCR Amplification
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DNA
(0.1-1.0 ug)
Single molecule array
Library
preparation Cluster generation 5’
5’ 3’
G
T
C
A
G
T
C
A
G
T
C
A
C
A
G
T
C
A
T
C
A
C
C
T
A G
C G
T
A
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Sequencing
Illumina Sequencing Technology
Sequencing By Synthesis (SBS) Technology
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TruSeq Chemistry: Flow Cell
Surface of flow
cell coated
with a lawn of
oligo pairs
8
channels
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1.6 Billion Clusters
Per Flow Cell
20
Microns
100
Microns
Sequencing
21
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100
Microns
Sequencing
22
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Patterned Flowcell
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Hiseq 3000: 478 million nanowells per lane
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What will go wrong ?
cluster identification
bubbles
synthesis errors:
Data Analysis
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What will go wrong ?
synthesis errors:
Phasing & Pre-Phasing problems
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HS2500 – PE250 - Q20
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Intensities - HS3K PE100
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HS3K PE150
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FASTQC
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FASTQC - Nextera
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FASTQC
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FASTQC
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FASTQC
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FASTQC
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FASTQC
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FASTQC
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FASTQC
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FASTQC
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“If you can put adapters on it, we can sequence it!”
single-stranded
Adapter
Ligation
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Optional: PCR-free libraries
PCR-free library:
– if concentration allows
– Reduction of PCR bias against e.g. GC rich or AT rich regions, especially for metagenomic samples
OR
Library enrichment by PCR:
– Ideal combination: high input and low cycle number; low-bias polymerase
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Quantitation & QC methods
Intercalating dye methods (PicoGreen, Qubit, etc.): Specific to dsDNA, accurate at low levels of DNA
Great for pooling of indexed libraries to be sequenced in one lane
Requires standard curve generation, many accurate pipetting steps
Bioanalyzer: Quantitation is good for rough estimate
Invaluable for library QC
High-sensitivity DNA chip allows quantitation of low DNA levels
qPCR Most accurate quantitation method
More labor-intensive
Must be compared to a control
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Library QC by Bioanalyzer
Predominant species of appropriate MW
Minimal primer dimer or adapter dimers
Minimal higher MW material
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Library QC by Bioanalyzer
Beautiful 100% Adapters
Beautiful
~ 125 bp
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Library QC
Examples for successful libraries Adapter
contamination
at ~125 bp
~125
bp
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Recommended RNA input
Library prep kit Starting material
mRNA (TruSeq) 100 ng – 4 μg total RNA
Directional mRNA (TruSeq) 1 – 5 μg total RNA or 50 ng mRNA
Apollo324 library robot
(strand specific)
100 ng mRNA
Small RNA (TruSeq) 1 μg total RNA
Ribo depletion (Epicentre) 1 – 5 μg total RNA
SMARTer™ Ultra Low RNA
(Clontech)
100 pg – 10 ng
Ovation RNA seq V2,
Single Cell RNA seq
(NuGen)
10 ng – 100 ng
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Standard RNA-Seq library protocol
QC of total RNA to assess integrity
Removal of rRNA (most common)
mRNA isolation
rRNA depletion
Fragmentation of RNA
Reverse transcription and second-
strand cDNA synthesis
Ligation of adapters
PCR Amplification
Purify, QC and Quantify
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• 18S (2500b) , 28S (4000b)
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RNA integrity <> reproducibility
Chen et al. 2014
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Considerations in choosing an RNA-Seq method
• Transcript type: - mRNA, extent of degradation - small/micro RNA • Strandedness: - un-directional ds cDNA library - directional library • Input RNA amount: - 0.1-4ug original total RNA - linear amplification from 0.5-10ng RNA • Complexity: - original abundance - cDNA normalization for uniformity • Boundary of transcripts: - identify 5’ and/or 3’ ends - poly-adenylation sites - Degradation, cleavage sites
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Is strand-specific information important?
Standard library
(non-directional)
antisense
sense
Neu1
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Strand-specific RNA-seq
Standard library (non-directional)
Antisense non-coding RNA
Sense transcripts
Informative for non-coding RNAs and antisense transcripts
Essential when NOT using polyA selection (mRNA)
No disadvantage to preserving strand specificity
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RNA-seq for DGE • Differential Gene Expression (DGE)
– 50 bp single end reads
– 30 million reads per sample (eukaryotes)
• 10 mill. reads > 80% of annotated genes
• 30 mill. . reads > 90% of annotated genes
– 10 million reads per sample (bacteria)
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Molecular indexing – for precision counts
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Molecular indexing – for precision counts
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Other RNA-seq • Transcriptome assembly:
– 300 bp paired end plus
– 100 bp paired end
• Long non coding RNA studies:
– 100 bp paired end
– 60-100 million reads
• Splice variant studies:
– 100 bp paired end
– 60-100 million reads
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RNA-seq – cheap and dirty
- 3’ Tag-sequencing
- Micro-array-like data
- Quant-Seq (coming soon)
- Brad-Seq (Townsley 2015)
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RNA-seq targeted sequencing:
- Capture-seq (Mercer et al. 2014) - Nimblegen and Illumina - Low quality DNA (FFPE) - Lower read numbers 10 million reads - Targeting lowly expressed genes.
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RNA-seq reproducibility
• Two big studies multi-center studies (2014)
• High reproducibility of data given: - same library prep kits, same protocols - same RNA–samples - RNA isolation protocols have to be identical - robotic library preps?
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Single Molecule Real Time (SMRT™) sequencing Sequencing of single DNA molecule by single polymerase Very long reads: average reads over 8 kb, up to 30 kb High error rate (~13%). Complementary to short accurate reads of Illumina
http://pacificbiosciences.com
THIRD GENERATION
DNA SEQUENCING
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70 nm aperture
“Zero Mode
Waveguide”
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Damien Peltier
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Paul Hagerman, Biochemistry and Molecular
Medicine, SOM. • Single-molecule sequencing of pure CGG array,
- first for disease-relevant allele. Loomis et al. (2012)
Genome Research.
- applicable to many other tandem repeat disorders.
• Direct genomic DNA sequencing of methyl groups,
- direct epigenetic sequencing (paper under review).
• Discovered 100% bias toward methylation of 20 CGG-
repeat allele in female,
– first direct methylated DNA sequencing in human
disease.
• DoD STTR award with PacBio. Basis of R01
applications.
First Sequencing of CGG-repeat Alleles in Human Fragile X
Syndrome using PacBio RS Sequencer
Nucleotide position
CGG36 CGG95
A
C
G
T
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Iso-Seq Pacbio
• Sequence full length transcripts no assembly
• High accuracy (except very long transcripts)
• More than 95% of genes show alternate splicing
• On average more than 5 isoforms/gene
• Precise delineation of transcript isoforms ( PCR artifacts? chimeras?)
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C1 Single cell capture
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10X Genomics
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25x linked –read coverage
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• 60 kb deletion
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Thank you!