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Deep Sequencing technologies
Gabriela Salinas
30 October 2017
Transcriptome and Genome Analysis Laboratory
Microarray and Deep-Sequencing Core Facility
University Medical Center Göttingen (UMG)
NGS-Course Molecular biology/Master
http://www.uni-bc.gwdg.de/index.php?id=709
Outline
➢ Deep Sequencing Technologies and Applications
➢ Second Generation Sequencing: Illumina platform
➢ Workflow: Library preparation
➢ TruSeq RNA-Standard library preparation (mRNA-Seq, non directional)
➢ Nextera™ technology (RNA-Seq, single cell)
➢ Strand-specific library preparation (directional)
➢ Workflow: Clonal amplification
➢ Workflow: Sequencing by Synthesis (SBS)
➢ Data Analysis and QC
➢ Single-Molecule, Real Time (SMRT) Sequencing
➢ Single-Molecule, Oxford Nanopore Technologies
Deep Sequencing Platforms and Applications
Long Reads
No required amplification
Simple sample prep
Single molecule raw accuracy
Errors tend to be random
Less coveragerequired
Short Reads
Amplification errors and bias
Several enzymatic steps
Multi molecule raw accuracy
Errors tend to be systematic
More coveragerequired
Second vs third generation sequencing approach
Second Generation SequencingIllumina Platform and Applications
Deep Sequencing Instruments_HiSeq 4000/HiSeq 2500
MiSeq
HiSeq 2000/HiSeq 2500 and HiSeq 4000
Instrument Output Fragment length Run time Human Genome per run-30x coverage
MiSeq 4.5 Gb -12 Gb 2x50; 2x150; 2x250, 2x300
4 hs – 48 hs No- only small genomes or gene panels
HiSeq2500 HTM 600 Gb SR up to 1x 100PE up to 2x 100
2 – 11 days 5
HiSeq2500 rapid mode 150 Gb PE up to 2x 250 4 hs – 48 hs 1
HiSeq4000 1500 Gb SR up to 1x 150PE up to 2x 150
4 hs – 48 hs 12
Illumina Sequencing Systems_HiSeq 4000/HiSeq 2500/MiSeq
1
2
3
4
Library Preparation
Cluster Generation
Sequencingby Synthesis
Data Analysis
Stranded total RNASeq● Depletion rRNA; mtRNA● RNA Fragmentation● ssDNA and dsDNA synthesis
● Hybridize to flow cell● Perform bridge amplification
● SR 50 bp; 6GB; ca. 50 Mio Reads/sample● Base Calling
● Images/Intensities QC● Clonal density QC● Pass Filter Reads QC● FastQ QC
RNA-Sequencing: Workflow_HiSeq 4000/2500
● End Repair● Ligate Adapters● Library validation QC
Data pre processing
cBot
Perform sequencing
DNA/RNA extraction
Experimentator
Dataprocessing
Bioinformatic
Applications for Deep Sequencing Technologies
Our sequencing applications include:
➢ Re-sequencing or de-novo sequencing of entire genomes
➢ Methylation analysis by bisulfite sequencing or by immunoprecipitated DNA (MeDIP)
➢ Chromatin modifications or
➢ Immunoprecipitated DNA to study DNA-protein interactions (ChIP seq)
➢ Transcriptome sequencing (RNA seq)
➢ Exome enrichment Analysis (DNA seq)
➢ Genome sequencing to discover SNPs and chromosomal rearrangements (DNA seq)
➢ small RNA Analysis
miRNAmRNA/ncRNAsChIP
DNA RNA
GenomeSeq
Met-Seq
Exom-Seq
Exon
Library Preparation
Project Information –
➢ Number of Samples
➢ Standard Operational Procedures (SOPs) to isolate RNA and DNA
➢ Amount of start material / FFPE
➢ Type of Library Preparation
➢ Strategies to improve the dynamic range
➢ Quality controls to be included / Validation of libraries
➢ Multiplexing Plans
➢ Number of Flow Cell Lanes
➢ Number of Sequencing Cycles
➢ Data Analysis Plans
Meta-Data required for deep Sequencing
RNA-Seq Methods
Standardnon-directional mRNA-Seq
strand-specificTotal RNA-Seq
Nextera RNA-Seq
Single cell RNA-SeqFFPE RNA-Seq
RNA-Seq Methods
Amount of Total RNA 1000-100 ng 50 ng 10 ng 1 ng
Nextera √ √ ? ?
mRNA √ x x x
Stranded total RNA √ x x x
End Repairand
phosphorylate
dA addition
A
Ligation
Purify Ligation (2x)
Final library
TruSeq Standard library preparation
➢Standard (mRNA)ds cDNA from fragmented RNA
P
A
AA
P
P+PT
P
P
P
RNA (Total)
mRNA
scDNA Synthesis
dcDNA Synthesis
Fragmentationpriming
PCR
Purify PCR
Library validation
QC_Bioanalyzer+NanoDrop
2 x purification (beads)
purification (beads)
QC_Bioanalyzer+NanoDropPicogreen or QuantiFluor
Purification (Beads)
Nextera™ technology: transposase mediated RNA-seq libraries (Tn-RNA-seq)
In vitro transposition withappended transposon ends
➢ Low input of start material (dsDNA: 50 ng)
➢ No mechanical fragmentation (sonication) is required
➢ 1 day library preparation (Tagmentation)
➢ Applicable to all organisms
➢ Optimized PCR: Improved formulations with fewer cycles deliver reduced GC bias and lower error rates
Advantages of the Nextera™ technology protocol
RNA (Total)
rRNA Depletion
scDNA Synthesis
dcDNA Synthesis
Fragmentation
QC_Bioanalyzer+NanoDrop
purification (beads)
purification (beads)
purification (beads)
Adenylate 3' Ends
Ligate Adapters
Validation
Pool
PCRPurify PCR
purification (beads)
purification (beads)
RNA (Total)
scDNA Synthesis
dcDNA Synthesis
Fragmentation
Adenylate 3' Ends
Ligate Adapters
Validation
Pool
PCRPurify PCR
RNA (Total)
scDNA Synthesis
dcDNA Synthesis
QC_Bioanalyzer+NanoDrop
purification (beads)
Tagmentation
Validation
Pool
Limited PCRPurify PCR
total RNA mRNANextera
RNA_Seq: Nextera, Stranded total RNA and Standard libraries
mRNA
purification (beads)
➢ Maintaining strand orientation allows identification of antisense expression (important mediator for gene regulation)
➢ Identification of mRNA; ncRNA (lincRNAs; snRNAs; snoRNAs and others)
➢ Increase alignment efficiency
➢ Currently available for human, mouse and rat (Ribo-Zero)
➢ Compatible with low quality samples (FFPE)
Advantages of the TruSeq Stranded total RNA library preparation
COVARIS (NGS)/Sonication
Blue Pippin/Fragment size
Fragment Analyzer/DNARNA Quality and Quantity
Sample Preparation Workflow: additional equipment
Library quality control: Fragment Analyzer
Library quality control: Fragment Analyzer
Clonal Amplification
A. B. C. D.
Clustering by Clonal Amplification-cBot
Image from http://www.illumina.com
Sequencing by Synthesis (SBS)
A. B. C. D.
Clustering by Clonal Amplification-cBot
Image from http://www.illumina.com
Sequencing by Synthesis_SBS
Image from http://www.illumina.com
● Sequencing by synthesis (SBS)
● The sequencing chemistry uses 4reversible terminator-bound, fluorescently labeled nucleotides
● A single nucleotide is incorporated into each DNA fragment and the fluorescence from each cluster isvisualized and measured by taking an image of theflow cell after laser excitation
● After incorporation and imaging, the fluorescent dye is enzymatically cleaved to allow incorporation of next nucleotide during the next flow cycle
Image from http://www.illumina.com
The reads are filtered based on a CHASTITY score allocated to each base (CHASTITY score for cycle)
QC_Sequencing and Cluster density
Data Analysis and QC
Gene Expression: Microarray/RNA-SeqStatistics; Hierarchical & K-means clusters; Scatter plots; list of candidates; RNA counting; Mapping to a reference
sequencing; pathways; gene ontologies; links to NCBI
Initial ConsultationUser & TAL
Experimental DesignTimeline and Cost
Project initiationUser
Sample preparation Sample collecting
TAL NGSSample quality controlSample library constructionClonal AmplificationSequencing_Instrument Run
Data AnalysisTAL
Pipelines; Data preprocessing;Txt files; cel Files; FastaQ Files
Epigenetics: Methylation; Chip-SeqMapping; peak calling; UCSC Browser; Literature searches
Small RNA: Microarray/small RNA-SeqMapping; known miRNA; unknown miRNA; Expression and counts;
links to miRBASE; known gene interactions
ResequencingMapping; SNP calling; small insertion/deletion;
large insertion/deletion; Copy Number Variation; inversions
Deep Sequencing Data Analysis Services_TAL
FastQC_Sequencing ReportInput formatAny fastq file, for example:@HWI-EAS91_1_30788AAXX:7:21:1542:1758GTCAATTGTACTGGTCAATACTAAAAGAATAGGATCGCTCCTAGCATCTGGAGTCTCTATCACCTGAGCCCA+HWI-EAS91_1_30788AAXX:7:21:1542:1758hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh`hfhhVZSWehR
Single-Molecule, Real Time (SMRTTM) Sequencing:Technology Update and Recent Applications
Single-Molecule, Real Time DNA Sequencing (SMRT) is:
Highquality, intact DNA samples are crucial to the success of PacBio SMART sequencing experiments.
DNA input requirement will vary by template inert size and project design: for instance 500 bp/600 ng; 10 Kb/12 µg.
Sample fragmentation: COVARIS
Fragment Size: Pippin Blue
PCR-free Sample Preparation Workflow
HLA FullLength Gene Sequencing
Reads Now Cover Entire Genes (With Introns)
Detection of DNA Base Modifications by SMRT Sequencing
De Novo Characterization of Genome & Epigenome
Characterization of Splice Variants
Recent Applications
https://www.youtube.com/watch?v=NHCJ8PtYCFc
Single-Molecule, Oxford Nanopore Technologies
Single-Molecule, Oxford Nanopore Technologies
Single-Molecule, Oxford Nanopore Technologies
Highquality, intact DNA samples are crucial to the success of PacBio or Nanopore sequencing experiments.
DNA input requirement and insert size selection is variable and depend on the application
Sample fragmentation: COVARIS „G tube“ or Pippin Blue
For Nanopore a library conditioning step which attaches the motor protein is required
PCR-free Sample Preparation Workflow
STATS
Price: Less than $1,000
DNA Read Length: 70,000 base pairs
1) Drop the DNA sample on a chip
2) Unzip the DNA
3) Block the ion current
4) Determine the sequence
5) Check for errors
MinIon Device
https://www.youtube.com/watch?v=3UHw22hBpAk