DNA Sequencing Technologies

Data Analysis in Genome Biology

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Thomas Girke

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April 10, 2018

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Usually We Prefer to Sequence DNA

• DNA sequencing is more efficient because:
• DNA cloning and amplification is easy
• Availability of efficient enzymatic sequencing reactions
• Protein sequencing is much harder because
• No comparable cloning or amplification techniques available
• Limited availability of enzymatic sequencing techniques
• Chemical nature of proteins makes sequencing harder

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DNA Libraries

A DNA library consists of cloned DNA fragments that can represent the entire genome of an organism (genomic DNA library) or its transcriptome (cDNA library).

Genomic library

• Contains often entire DNA content of an organism.
• Suitable for determining genomic DNA sequence.
• Requires chromosomal DNA isolation.

cDNA library

• Contains the mRNAs that are expressed in a tissue sample.
• mRNA is used as starting material
• mRNA needs to be reverse transcribed into cDNA
• Requires mRNA isolation
• Challenges: cDNA libraries tend to be incomplete with regard to
• 5’ sequences
• Representation of all genes in the genome

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Why Is it Helpful to Have Both?

• Genomic library gives genome sequence
• cDNA library gives information about expressed sequences in genome
• For instance: cDNA sequences can be aligned to newly generated genomic sequence to
• identify gene boundaries ⇒ gene prediction with physical evidence
• distinguish between expressed genes and pseudo-genes
• Many other reasons

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Workflow of a Genomic Sequencing Project

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Annotation of Functional Features

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Submit to GenBank

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Synthesis of Common Genomic Libraries

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Plasmid Library λ Phage Library

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Synthesis of cDNA Library in λ Phage Vector

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1. mRNA to cDNA 2. cDNA Cloning into λ

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Cloning Vectors for Libraries

Plasmid Library [ Genomic & cDNA ]

• Circular extra-chromosomal DNA molecule in bacteria
• Maximum insert size for cloning: 1,000-20,000 bp

λ Phage Library [ Genomic & cDNA ]

• Double-stranded linear DNA of E. coli infecting virus
• Maximum insert size for cloning: 1,000-25,000 bp

Cosmid Library [ Genomic ]

• λ phage-derived hybrid plasmid with cos sequences
• Maximum insert size for cloning: 35,000 to 50,000 bp

BAC Library [ Genomic ]

• Bacterial artificial chromosome
• Maximum insert size for cloning: 150,000-350,000 bp

YAC Library [ Genomic ]

• Yeast artificial chromosome
• Maximum insert size for cloning: 100,000-3,000,000 bp

Many Additional Library Types

• Please consult molecular biology books.

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What are EST Sequences?

• Expressed sequence tags or ESTs are short single-pass sequences from cDNA libraries (partial RNA sequences).
• They are useful for:
• Identification of gene boundaries in genomic sequences
• Discovery of single nucleotide polymorphisms (SNPs)
• Discovery of alternative splice events in transcripts
• Quantitative (digital) gene expression analysis (DGE)
• Currently, there are hundreds of million ESTs available in GenBank
• RNA-Seq reads are the NGS version of ESTs
• Many additional applications.

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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History of DNA Sequencing

1977. ”DNA Sequencing by Chemical Degradation” is published by Allan Maxam and Walter Gilbert.
1977. ”DNA Sequencing by Enzymatic Synthesis” is published by Fred Sanger.
1980. Fred Sanger and Walter Gilbert receive the Nobel Prize in Chemistry.
1982. GenBank starts as a public repository of DNA sequences.
1986. Leroy Hood’s laboratory at the California Institute of Technology announces the first semi-automated DNA sequencing machine.
1997. Genome sequence of E. coli is published.
2001. Draft sequence of the Human genome is published.
2004. First next generation sequencing technologies become available to the public.

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Chemical Sequencing by Maxam & Gilbert

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1. Uses radioactive labeled DNA fragments of 500 bp.
2. Four separate chemical treatments generate DNA breaks at the positions: G, A+G, C, C+T.
3. The fragments are size-separated by gel electrophoresis in four separate lanes.
4. Visualization of the fragments by autoradiography on an X-ray film.

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Chemical DNA Degradation Gel Electrophoresis

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Illustration of Sanger Sequencing

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Sequencing Principle

Radioactive Fluorescence

Labeling

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Processing of Sequencing Raw Data

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• Assign quality score to each peak
• The frequently used Phred scores provide log(10)-transformed error probability values:
• score = 20 corresponds to a 1% error rate
• score = 30 corresponds to a 0.1% error rate
• score = 40 corresponds to a 0.01% error rate
• The base calling (A, T, G or C) is performed based on Phred scores.
• Ambiguous positions with Phred scores ≤ 20 are labeled with N.

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Common Synonyms

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• High-throughput sequencing: HTS or HT-Seq
• Flow cell sequencing (FCS)
• Massively parallel sequencing (MPS)
• Next generation sequencing (NGS)
• Second/third generation sequencing
• Deep sequencing
• Sequencing by synthesis
• Many other synonyms
• Review article: Holt et al 2008

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Overview: 454, SOLiD and Illumina

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From review article: Medini et al 2008

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Similarities and Differences of NGS Technologies

Common components

• Flow cells as reaction chambers
• Iterative sequencing process
• Massive parallelization
• Clonally amplified or single molecule templates

Differences

• Template preparation
• Sequencing chemistry
• Flow cell configuration

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NGS Sequencing Methods

Reversible Terminator Methods (e.g. Illumina/Solexa)

• Use reversible versions of dye-terminator reactions.
• Principle steps: adding one nucleotide at a time, detecting fluorescence corresponding to that position, then removing the blocking group to allow polymerization of another nucleotide.

Single Molecule Methods (e.g. Helicos, PacBio)

• Variable strategies.

Pyrosequencing Methods (e.g. 454)

• Also use DNA polymerization to add nucleotides.
• Principle steps: adding one type of nucleotide at a time, then detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.

Supported Oligonucleotide Ligation Methods (e.g. SOLiD, Complete Genomics)

• Uses ligation-based approach
• Principle steps: stepwise ligation of labeled random octamers to obtain sequence of attached dinucleotides; the ligated dinucleotides of each ligation round are spaced by several nucleotides; continuous sequence information is obtained by offsetting sequencing primer.

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Example: Illumina/Solexa Technology

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Illumina HiSeq 2500 Sequencer

Flow Cell

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Basic Steps of Illumina Sequencing

Flow Cell Loading

1. Generate DNA library (genomic- or cDNA-based) with insert length of ∼200 bp.
2. Load library onto flow cell (nano device for liquid handling).
3. PCR-based bridge amplification of loaded fragments to obtain DNA clusters (serves signal amplification)

Sequencing Cycles

4. Start reversible dye-terminator reaction containing primer and labeled dNTPs among other components.
5. Image scan to detect the identity of first base of each cluster via the characteristic fluorescence signal for each labeled nucleotide.
6. De-protection step removes the blocking group and fluorescence group of the incorporated nucleotide.
7. Repeat steps 4-6 about 50-250 times.

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Compare with illustration on next 3 slides!

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Flow Cell Loading

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Illumina HiSeq 2500 Sequencer

Flow Cell

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Sequencing Cycles

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Details of Sequencing Reaction

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Illustration shows the sequencing cycles for a single template molecule!

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Single End, Paired End and Mate Pair Sequencing

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Single End

Paired End

Mate Pair

AP1/AP2: flow cell adapators; SP1/SP2: sequencing primers

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Paired End Chemistry: Step I

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Single End

Paired End

Grafted Flow Cell

Cluster Generation: Initial Extension

Linearization: periodate two different enzymes

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Paired End Chemistry: Step II

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Cluster Generation: Amplification

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Paired End Chemistry: Step III

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Cluster Generation: Linearization

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Paired End Chemistry: Step IV

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Sequencing

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Processing of Illumina Sequencing Data

• Convert cluster images to intensity values.
• Base calling based on intensity for each fluorescence dye.
• Generates quality scores similar to Phred scores.
• The length of each sequence corresponds to the number of cycles, e.g. 75 cycles → 75 bp.
• Remove sequences with low quality reads.
• Downstream analyses specific to application!

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A single sequencing run with 2x 100 cycles can generate ∼ 3 billion sequences and 32TB of image data.

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Sequence Format: FASTQ

@SRR446037.238 length=75

TCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTTGATTTCTCATAAGGTGCCAGCGGAGTCCTATAAG

+SRR446037.238 length=75

IIIIIIIIIIIGIIHIHIIIIIIIIDHDIIIIIFHDGDEFHCCGHHHHHCDDD@?6?@A@?A;??@2@BA@BBB@

... millions of entries ...

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FASTQ format has 4 lines per sequence

1. '@' character followed by a sequence identifier
2. Character string of sequence
3. '+' character optionally followed by the same sequence identifier
4. Quality scores in ASCII format

Example of partial FASTQ file

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Helicos: Single Molecule Sequencing

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• Has similarities to Solexa/Illumina technology, but sequences single molecule templates.
• Attaches one of the four nucleotides at a time using proprietary nucleotide-polymerase formulations. This prevents the incorporation of more than one nucleotide in each cycle in homopolymer regions.

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

• ​

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454/Roche Sequencing Steps

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Pyrosequencing Methods (e.g. 454)

• Also uses DNA polymerization to add nucleotides.
• Principle steps: adding one type of nucleotide at a time, then detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.

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For more details see: 454 Web Site (http://www.454.com)

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454 Pyrosequencing

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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SOLiD: Sequencing by Supported Oligonucleotide

Ligation and Detection

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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PacBio: Sequencing with Single Polymerase Molecule

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Publication: Eid et al 2009

• Read lengths: 0.5-20kbp!!
• Much lower cost
• Disadvantages: high error rate and low number of sequences

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Nanopore Sequencing

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• Read lengths: xkbp!!
• Much lower cost.
• Disadvantages: currently higher error rate and low number of sequences.

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Comparison of Methods

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All numbers are estimates and apply to the situation in Dec. 2015!

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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Applications of NGS Methods

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NGS technologies provide vast opportunities for genomics, comparative genome biology, medical diagnostics, etc. The following list only a few of examples.

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Applications

• Genome-wide detection of SNPs and mutations (SNP-seq)
• Methylome profiling by bisulphite sequencing (BS-seq)
• DNA-protein interactions (ChIP-seq)
• Transcriptome sequencing (RNA-seq)
• mRNA expression profiling (RNA-seq and DGE)
• Small RNA profiling and discovery
• Hi-C to study three dimensional architecture of genomes
• De novo genome assembly (for PacBio)

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Application: De Novo Sequencing and Assembly

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Application: DNA-Protein Interactions with ChIP-Seq

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Reference for ChIP-Seq data analysis: Jothi et al 2008

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Application: Methylome Profiling with BS-Seq

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Application: RNA-Seq Gene Expression Profiling

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Application: Digital Gene Expression (DGE) Profiling

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Sequencing

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Targeted Sequencing for Large Genomes

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Targeted sequencing using DNA capture microarrays or beads

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• Powerful approach to make NGS-Seq more economic and versatile.
• Example: usage of programmable microarrays (here NimbleGen) to enrich for DNA regions of interest (Albert et al 2007).

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10X Genomics: Linked-Read Sequencing

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Resolves many challenges inherent to short read sequencing

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From Zheng et al (2016)

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Database: Sequence Read Archive from NCBI

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• SRA: http://www.ncbi.nlm.nih.gov/Traces/sra

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• 1000 Human Genomes Project: http://www.1000genomes.org

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• Many more 1000 genome projects

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Outline

What Are We Sequencing?

• Genomic Libraries
• cDNA Libraries

Traditional DNA Sequencing Technologies

• Chemical Sequencing
• Sanger Sequencing

Next Generation Sequencing Methods

• Solexa/Illumina: Reversible Terminator Method
• Helicos: Single Molecule Sequencing
• 454/Roche: Pyrosequencing Method
• SOLiD/ABI: Supported Oligo Ligation Method
• Third Generation Sequencing: PacBio and Others

Research Applications

References and Books

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References

Albert, T J, Molla, M N, Muzny, D M, Nazareth, L, Wheeler, D, Song, X, Richmond, T A, Middle, C M, Rodesch, M J, Packard, C J, Weinstock, G M, Gibbs, R A (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods, 4: 903-905. URL http://www.hubmed.org/display.cgi?uids=17934467

Eid, J et al. (2009) Real-time DNA sequencing from single polymerase molecules. Science, 323: 133-138. URL http://www.hubmed.org/display.cgi?uids=19023044

Holt, RA, Jones, SJ (2008) The new paradigm of flow cell sequencing. Genome Res, 18: 839-846. URL http://www.hubmed.org/display.cgi?uids=18519653

Jothi, R, Cuddapah, S, Barski, A, Cui, K, Zhao, K (2008) Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data. Nucleic Acids Res, 36: 5221-5231. URL http://www.hubmed.org/display.cgi?uids=18684996

Medini, D, Serruto, D, Parkhill, J, … , C, Moxon, R, Falkow, S, Rappuoli, R (2008) Microbiology in the post-genomic era. Nat Rev Microbiol, 6: 419-430. URL http://www.hubmed.org/display.cgi?uids=18475305

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References

Zheng GXY et al (2016) Haplotyping germline and cancer genomes with high-throughput linked-read sequencing. Nat Biotechnol 34: 303–311

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