Next generation sequencing
- 1. Next generation sequencing By Neelma Nayab MPHIL 2ND SEMESTER BT320192021 Department of Biotechnology and Genetic Engineering Kohat University of Science and Technology, Kohat 26000 Khyber Pakhtunkhwa, Pakistan June 2020
- 2. WHAT IS SEQUENCING? Genome sequencing is a method used to figure out the order of DNA nucleotides, or bases in a genome-the order of A, C, G, and T that make up an organism's DNA. In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be determined. Next-generation sequencing techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing.
- 3. IMPORTANT OF DNA SEQUENCING: Better understanding of gene expression. Gene expression has significance in protein creation etc. It is capable detect various diseases and genetic illnesses. Personalized medicine and disease discovery is possible. Forensics NEXET GENERATION SEQUENCING: Next-generation sequencing (NGS), also known as high- throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies including: ILLUMINA (SOLEXA) SEQUENCING ROCHE 454 SEQUENCING (PYROSEQUENCING) SOLID SEQUENCING ION TORRENT: PROTON / PGM SEQUENCING These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such have revolutionized the study of genomics and molecular biology. NGS has brought high speed not only to genome sequencing and personal medicine it has also changed the way we do genome research. OVERVIEW OF NEXT GENERATION SEQUENCING PROTOCOL
- 4. (Fig1) 1 ILLUMINA (SOLEXA) SEQUENCING Illumina Genome Analyzer is a high-throughput, short-read, massively parallel sequencing platform. The Illumina Solexa sequencing technology uses sequencing-by-synthesis on an eight-channel flowcell to produce more than 10 million reads per channel with read lengths up to 100bp. Individual fragments of a genomic DNA library are amplified on
- 5. a flowcell via bridge-PCR to generate clusters of identical fragments. Reversible terminator nucleotides are used in sequencing allowing for the reading of one base per sequencing cycle per cluster. This platform enables many applications, including whole genome resequencing, transcriptome sequencing, gene expression profiling, and epigenomic sequencing. How does Illumina DNA sequencing work? 1. The first step in this sequencing technique is to break up the DNA ?into more manageable fragments of around 200 to 600 base pairs?. 2. Short sequences of DNA called adaptors?, are attached to the DNA fragments. 3. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide. 4. Once prepared, the DNA fragments are washed across the flowcell. The complementary DNA binds to primers? on the surface of the flowcell and DNA that doesn’t attach is washed away. 5. The DNA attached to the flowcell is then replicated to form small clusters of DNA with the same sequence. When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera. 6. Unlabelled nucleotide bases? and DNA polymerase? are then added to lengthen and join the strands of DNA attached to the flowcell. This creates ‘bridges’ of double-stranded DNA between the primers on the flowcell surface. 7. The double-stranded DNA is then broken down into single- stranded DNA using heat, leaving several million dense clusters of identical DNA sequences. 8. Primers and fluorescently?-labelled terminators (terminators are a version of nucleotide base – A, C, G or T - that stop DNA synthesis) are added to the flowcell. 9. The primer attaches to the DNA being sequenced.
- 6. 10. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA. 11. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base. This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G and T) give off a different colour. 12. The fluorescently-labelled terminator group is then removed from the first base and the next fluorescently-labelled terminator base can be added alongside. And so the process continues until millions of clusters have been sequenced. 13. The DNA sequence is analysed base-by-base during Illumina sequencing, making it a highly accurate method. The sequence generated can then be aligned to a reference sequence, this looks for matches or changes in the sequenced DNA.(fig 2,3)
- 7. (Fig 2)
- 8. (Fig 3) 2 ROCHE 454 SEQUENCING (PYROSEQUENCING) A method of DNA sequencing based on the “sequencing by synthesis" principle. It differs from Sanger sequencing, relying on the detection of pyrophosphate release (hence the name) on nucleotide incorporation, rather than chain termination with dideoxynucleotides.
- 9. ssDNA template is hybridized to a sequencing primer Incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine5´phosphosulfate (APS) and luciferin. PYROSEQUENCING CHEMISTRY:
- 10. PYRO SEQUENCING PYROSEQUENCING PROTOCOL: (Fig 4) The addition of one of the fourdeoxynucleotide triphosphates (dNTPs)(in the case ofdATP we add dATPαS which is not a substrate for a luciferase) initiates thesecond step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate.
- 11. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide. 3 SOLiD SEQUENCING SOLiD is an enzymatic method of sequencing that uses DNA ligase, an enzyme used widely in biotechnology for its ability to ligate double- stranded DNA strands . Emulsion PCR is used to immobilise/amplify a ssDNA primer-binding region (known as an adapter) which has been conjugated to the target sequence (i.e. the sequence that is to be sequenced) on a bead. These beads are then deposited onto a glass surface − a high density of beads can be achieved which which in turn, increases the throughput of the technique.(Fig 5)
- 12. (Fig 5)