Next generation sequences
Download as PPTX, PDF0 likes422 views
This document discusses next generation DNA sequencing technologies. It describes several second generation sequencing technologies including ABI/SOLiD sequencing, Ion Torrent sequencing, and Oxford Nanopore sequencing. ABI/SOLiD sequencing uses DNA ligase and fluorescent labels to sequence DNA in a cyclic ligation process. Ion Torrent sequencing detects hydrogen ions released during DNA polymerization to determine sequences. Oxford Nanopore sequencing analyzes variations in ionic current as single-stranded DNA passes through a nanopore to sequence DNA. These next generation techniques enable massively parallel sequencing and longer reads compared to first generation methods.
Next generation sequences
- 1. NEXT GENERATION SEQUENCES Bhanu Krishan MSc. Biotechnology, Sem III GGDSD College, Chandigarh
- 2. Topics Included • Role of DNA Sequencing • NEXT GENERATION SEQUENCING • Second Generation Sequencers • Third Generation Sequencer
- 3. Role of DNA Sequencing • DNA sequencing is the discovery that uses the DNA composition to understand and decrypt the code to all biological life on earth as well as to understand and treat genetic diseases. • The sequencing technologies has played an important role in the analysis of genomic sequences of any organisms. • A DNA sequencer produces files containing DNA sequences. • These sequences are strings called reads on an alphabet formed by five letters {A, T, C, G, N}. The symbol N is used to represent an ambiguity.
- 4. NEXT GENERATION SEQUENCING TECHNOLOGIES • Known under the name of “next generation sequencing (NGS) technologies” or “high throughput sequencing technologies. • NGS technologies can sequence parallel millions to billions of reads in a single run. • The time required to generate the gigabase sized reads is only a few days or hours • Literature divided NGS technologies into two types: Second generation sequencing technologies which refer to the newest sequencing technologies developed in the NGS environment after thefirst generation Third generation sequencing technologies that are sequencing technologies recently appeared.
- 6. ABI/ SOLiD Sequencing • Supported Oligonucleotide Ligation and Detection (SOLiD) is a NGS sequencer Marketed by Life Technologies. • SOLiD is an enzymatic method of sequencing that uses DNA ligase. • The ABI/SOLiD process consists of multiple sequencing rounds It starts by attaching adapters to the DNA fragments, fixed on beads and cloned by PCR emulsion. These beads are then placed on a glass slide and the 8-mer with a fluorescent label at the end are sequentially ligated to DNA fragments, and the color emitted by the label is recorded.
- 7. Then, the output format is color space which is the encoded form of the nucleotide where four fluorescent colors are used to represent 16 possible combinations of two bases. A bases and the sequence of the DNA fragment can be deduced. The sequencer repeats this ligation cycle and each cycle the complementary strand is removed, and a new sequencing cycle starts at the position n-1 of the template. The cycle is repeated until each base is sequenced twice. The recovered data from the color space can be translated to letters of DNA bases and the sequence of the DNA fragment can be deduced. The strength of ABI/ SOLiD platform is high accuracy because each base is read twice while the drawback is the relatively short reads and long run times. The errors of sequencing in this technology is due to noise during the ligation cycle which causes error identification of bases.
- 8. Figure 2: SUPPORTOLIGONUCLEOTIDE LIGA TION DETECTION
- 10. Figure 4: ABI/ SOLiD sequencer, Source: Biogene Blog
- 11. Ion Torrent Sequencing Life Technologies commercialized the Ion Torrent semiconductor sequencing technology in 2010. Similar to Roche/454 pyrosequencing technology but it does not use fluorescent labeled nucleotides like other second-generation technologies. Based on the detection of the hydrogen ion released during the sequencing process. Uses a chip that contains a set of micro wells and each has a bead with several identical fragments
- 12. The incorporation of each nucleotide with a fragment in the pearl, a hydrogen ion is released which change the pH of the solution. This change is detected by a sensor attached to the bottom of the micro well and converted into a voltage signal which is proportional to the number of nucleotides incorporated. The Ion Torrent sequencers are capable of producing reads lengths of 200 bp, 400 bp and 600 bp with throughput that can reach 10 Gb for ion proton sequencer. Figure 5: Ion Torrent Sequencer, Source: Camlin | Intelligent Technology
- 13. Ion Torrent Sequencing Advantages : Focused on read lengths which are longer to other SGS sequencers Fast sequencing time between 2 and 8 hours. Disadvantage: Difficulty of interpreting the homopolymer sequences (more than 6 bp) which causes insertion and deletion (indel) error with a rate about ~1%.
- 14. Figure 6: Ion Torrent, After the introduction of a single nucleotide species, the unincorporated bases are washed away and the next is added Semiconductor sequencing As a base is incorporated, a single H+ ion is released, which is detected by a CMOS–ISFET sensor Single nucleotide addition Only one dNTP species is present during each cycle; several identical dNTPs can be incorporated during a cycle, increasing the emitted ions
- 15. Oxford Nanopore Sequencing • The Oxford Nanopore sequencing (ONT) was developed as a technique to determine the order of nucleotides in a DNA sequence. • In 2014, Oxford Nanopore Technologies released the MinION device that promises to generate longer reads that will ensure a better resolution structural genomic variants and repeat content. • It’s a mobile single-molecule Nanopore sequencing measures four inches in length and is connected by a USB 3.0 port of a laptop computer. Figure 7: Oxford Nanopore MinION.
- 16. • In this sequencing technology, the first strand of a DNA molecule is linked by a hairpin to its complementary strand. • The DNA fragment is passed through a protein nanopore (a nanopore is a nanoscale hole made of proteins or synthetic materials). • When the DNA fragment is translated through the pore by the action of a motor protein attached to the pore, it generates a variation of an ionic current caused by differences in the moving nucleotides occupying the pore • This variation of ionic current is recorded progressively on a graphic model and then interpreted to identify the sequence • The sequencing is made on the direct strand generating the “template read” and then the hairpin structure is read followed by the inverse strand generating the “complement read”, these reads is called "1D". • If the “temple” and “complement” reads are combined, then we have a resulting consensus sequence called “two direction read” or "2D".
- 17. Figure 8: The Oxford Nanopore sequencing process. (A) Suspended library molecules are concentrated near nanopores embedded in the membrane. A voltage applied across the membrane induces a current through the nanopores. (B) Schematic of a library molecule, showing dsDNA ligated to a leader adapter pre-loaded with a motor protein and a hairpin adapter pre-loaded with a hairpin protein, and the tethering oligos. (C) Sequencing starts from the 5' end of the leader adapter. The motor protein unwinds the dsDNA allowing single-stranded DNA to pass through the pore. (D) A flow cell contains 512 channels (grey), each channel consisting of 4 wells (white). Each well contains a pore (blue) and a sensor. At any given time, the device is recording the data stream from the wells of the active well- group, in this example, g1. (E) Perturbation in the current across the nanopore is measured 3,000 times per second as ssDNA passes through the nanopore. (F) The 'bulk data' are segmented into discrete 'events' of similar consecutive measurements. The 5-mer corresponding to each event is inferred using a statistical model. (G) The 1D base-calls are inferred separately for the template and complement event signals. (H) Alignment of the 2D base-calls from the event signals from both, and the 1D base-calls are used to constrain the 2D base-calls.
- 18. • Advantages offered by MiniION sequencer: • Low cost and small size. • Sample is loaded into a port on the device and data is displayed on the screen and generated without having to wait till the run is complete. • Provide very long reads exceeding 150kbp which can improve the contiguity of the de-novo assembly
- 19. References • Kchouk M, Gibrat JF, Elloumi M. Generations of sequencing technologies: from first to next generation. Biology and Medicine. 2017;9(3). • Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nature Reviews Genetics. 2016 Jun;17(6):333-51. • Mardis ER. Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet.. 2008 Sep 22;9:387-402. • Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, Hoon J. An integrated semiconductor device enabling non-optical genome sequencing. Nature. 2011 Jul;475(7356):348-52. • Mikheyev AS, Tin MM. A first look at the Oxford Nanopore MinION sequencer. Molecular ecology resources. 2014 Nov;14(6):1097-102. • Laehnemann D, Borkhardt A, McHardy AC. Denoising DNA deep sequencing data—high- throughput sequencing errors and their correction. Briefings in bioinformatics. 2016 Jan 1;17(1):154-79.