DNA Sequencing: Next Generation Sequencing (NGS) Lecture PDF

DNA Sequencing: Next Generation Sequencing (NGS) RDNA202 Cassie [email protected]  Human Genome Project The first major foray into DNA sequencing = Human Genome Project Used first-generation sequencing – Sanger Sequencing (Chain Termination method) Took 13 ye...

DNA Sequencing: Next Generation Sequencing (NGS) RDNA202 Cassie [email protected]  Human Genome Project The first major foray into DNA sequencing = Human Genome Project Used first-generation sequencing – Sanger Sequencing (Chain Termination method) Took 13 years Cost $3 billion Completed in 2003  Next Generation Sequencing (NGS) “Next generation” implies next step in development of DNA sequencing technology Second-Generation Sequencing Introduced in 2004 & 2006 Provides High-throughput sequencing  Next Generation Sequencing (NGS) Massively parallel sequencing technology Offers ultra-high throughput, scalability, and speed. Used to determine order of nucleotides: In entire genomes Targeted regions of DNA or RNA Has revolutionized biological sciences – allows wide variety of applications  Next Generation Sequencing (NGS) Biggest advances in genome sequencing Due to increased speed and accuracy Results in decreased cost and manpower requirements NGS has decreased cost per megabase Increased number and diversity of sequenced genomes https://www.yourgenome.o rg  Applications of NGS Rapidly sequence whole genomes Deeply sequence target regions Utilise RNA sequencing (RNA-seq) to discover: Novel RNA variants and splice sites Quantify mRNAs for gene expression analysis Analyse epigenetic factors Genome-wide DNA methylation DNA-protein interactions Sequence cancer samples – study rare somatic variants, tumour subclones, etc. Identify novel pathogens  Categories of NGS Two Major categories 1. Sequencing by hybridization Uses specific probes to interrogate sequences Used in diagnostic applications for: Identifying disease-related SNPs Identifying gross chromosome abnormalities – rearrangements, deletions, duplications, copy number variants (CNVs) 2. Sequencing by Synthesis (SBS) Further development of Sanger sequencing – without the ddNTPs Uses combination of repeated synthesis cycles & methods to incorporate nucleotides into growing chain For interest – there is also Pyrosequencing and some others– Not going to go into detail in this section  Similarities between different NGS technologies 1. Sample Preparation Requires library obtained by: Amplification or ligation with custom adapter sequences Adapter sequences allow library hybridization to the sequencing chips Provide universal priming site for sequencing primers  Similarities between different NGS technologies 2. Sequencing machines Each library fragment is amplified on a solid surface – either beads or flat silicon derived surface This is done using covalently attached DNA linkers – hybridize library adapters Amplification creates clusters of DNA – each originating from single library fragment Each cluster acts as individual sequencing reaction The sequence from each cluster is optically read – either by light or fluorescence Each machine has own cycling condition. Similarities between different NGS technologies 3. Data output Each machine provides raw data at the end of sequencing run Raw data = collection of DNA sequences generated at each cluster This data is further analysed to provide meaningful results Sequencing by Synthesis (SBS) properties Relies on shorter reads (300-500 bp) Generally has intrinsically higher error rate relative to Sanger Due to incomplete removal of fluorescent signal which can cause higher background noise levels Relies on high sequence coverage – “massively parallel sequencing” Of millions to billions of short DNA sequence reads (50 – 300 nucleotides ) = Short Read Sequencing Sequencing by Synthesis (SBS) properties Utilises step-by-step incorporation of reversibly fluorescent and terminated nucleotides Nucleotides modified in two ways 1. Each nucleotide – reversibly attached to a single fluorescent molecule with unique emission wavelengths 2. Each nucleotide is also reversibly terminated - ensures only one nucleotide incorporated per cycle  Sequencing by Synthesis (SBS) Process All four nucleotides – added to sequencing chip Single nucleotide is incorporated into sequence Remaining nucleotides – washed away Fluorescent signal is read at each cluster and recorded Both Fluorescent molecule and terminator group are cleaved and washed away Process is repeated until sequencing is complete NGS technologies 454 sequencing or pyrosequencing (Roche Applied Science) Solexa Technology (Used in Illumina genome analyzer) The SOLiD platform (Applied biosystems) Ion Torrent: Proton/PGM sequencing The HeliScope Single Molecule Sequencer Technology SMRT Pacific Biosciences Illumina (Solexa) Sequencing – Overview (Cyclic Reversible Termination) Shin et al. 2014  Illumina Sequencing – Step 1: Library preparation First Step – break up DNA into more manageable fragments (~200 – 600bp) Short sequences of DNA – adapters – attached to DNA fragments DNA fragments attached to adapters – denatured (made single stranded) Libraries constructed to give mixture of adapter-flanked fragments up to several hundred bp in length Illumina Sequencing – Step 1: Library preparation Summary 1. DNA fragmentation 2. Adaptor ligation 3. Library quantitation  Illumina Sequencing – Step 2: Bridge amplification Illumina technology relies on bridge PCR to amplify genomic region that needs to be sequenced An in vitro constructed adapter flanked library is PCR amplified But both primers densely coat surface of solid substate – attached at their 5’ end by a flexible linker Because of this – amplification products from template library – remain locally attached near point of origin  Illumina Sequencing – Step 2: Bridge amplification At the end of the PCR – each clonal cluster contains ~1000 copies of a single member of the template library DNA fragments attached to adapters – are then made single-stranded Once prepared – DNA fragments are washed across the flow cell (also known as the sequencing chip) Complementary DNA binds to primers on surface of flow cell DNA that doesn’t attach is washed away Illumina Sequencing – Step 2: Bridge amplification 1. Complementary strand of DNA fragment in library is synthesised 2. Complementary strand folds over and anneals with the other type of flow cell oligo – forms a bridge 3. Double stranded bridge – denatured – forming two single strands attached to the flow cell 4. Process of bridge amplification repeats 5. More clones of double stranded bridges formed  Illumina Sequencing – Step 2: Bridge amplification – Clonal clustering Double-stranded clonal bridges are denatured Reverse strands are removed Forward strands remain as clusters for sequencing Illumina Sequencing – Step 3: Sequencing Reaction Bridge amplification – Clonal clustering Components 1. Primers 2. dNTPs Labelled with a fluorescent dye Contains a reversible terminator (Trinitrogen = N3) 3. DNA polymerase  Illumina Sequencing – Step 3: Sequencing Reaction Bridge amplification – Clonal clustering Process Cyclic reversible termination Only one of four fluorescent dNTPs added per cycles Images of clusters – captured after incorporation of each nucleotide After imaging, fluorescent dye and terminator are cleaved and released Illumina Sequencing – Step 3 Trinitrogen (N3) Illumina Sequencing – Step 3 Illumina Sequencin g: Overview  When to use NGS vs Sanger Sequencing Sanger sequencing: Good choice when investigating a small region of DNA on limited number of samples or genomic targets (~20 or fewer) NGS Allows you to screen more samples cost-effectively Allows detection of multiple variants across targeted areas of the genome Used when Sanger sequencing approaches would be too costly and time-consuming