Sanger sequencing
Sanger sequencing is a method of
Method
The classical chain-termination method requires a single-stranded DNA template, a DNA
The DNA sample is divided into four separate sequencing reactions, containing all four of the standard
In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes, from bottom to top, are then used to read the DNA sequence.
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.
Dye-terminator sequencing
Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emits light at different wavelengths.
Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace
This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, was used for the vast majority of sequencing projects until the introduction of
Automation and sample preparation
Applications of dye-terminating sequencing
The field of public health plays many roles to support patient diagnostics as well as environmental surveillance of potential toxic substances and circulating biological pathogens. Public health laboratories (PHL) and other laboratories around the world have played a pivotal role in providing rapid sequencing data for the surveillance of the virus
Sanger sequencing is also the "gold standard" for norovirus surveillance methods for the Center for Disease Control and Prevention's (CDC) CaliciNet network. CalciNet is an outbreak surveillance network that was established in March 2009. The goal of the network is to collect sequencing data of circulating noroviruses in the United States and activate downstream action to determine the source of infection to mitigate the spread of the virus. The CalciNet network has identified many infections as foodborne illnesses.[3] This data can then be published and used to develop recommendations for future action to prevent tainting food. The methods employed for detection of norovirus involve targeted amplification of specific areas of the genome. The amplicons are then sequenced using dye-terminating Sanger sequencing and the chromatograms and sequences generated are analyzed with a software package developed in BioNumerics. Sequences are tracked and strain relatedness is studied to infer epidemiological relevance.
Challenges
Common challenges of DNA sequencing with the Sanger method include poor quality in the first 15-40 bases of the sequence due to primer binding and deteriorating quality of sequencing traces after 700-900 bases. Base calling software such as Phred typically provides an estimate of quality to aid in trimming of low-quality regions of sequences.[10][11]
In cases where DNA fragments are
Current methods can directly sequence only relatively short (300-1000
Microfluidic Sanger sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates long and accurate sequence reads, while obviating many of the significant shortcomings of the conventional Sanger method (e.g. high consumption of expensive reagents, reliance on expensive equipment, personnel-intensive manipulations, etc.) by integrating and automating the Sanger sequencing steps.
In its modern inception, high-throughput genome sequencing involves fragmenting the genome into small single-stranded pieces, followed by amplification of the fragments by polymerase chain reaction (PCR). Adopting the Sanger method, each DNA fragment is irreversibly terminated with the incorporation of a fluorescently labeled dideoxy chain-terminating nucleotide, thereby producing a DNA “ladder” of fragments that each differ in length by one base and bear a base-specific fluorescent label at the terminal base. Amplified base ladders are then separated by capillary array electrophoresis (CAE) with automated, in situ “finish-line” detection of the fluorescently labeled ssDNA fragments, which provides an ordered sequence of the fragments. These sequence reads are then computer assembled into overlapping or contiguous sequences (termed "contigs") which resemble the full genomic sequence once fully assembled.[14]
Sanger methods achieve maximum read lengths of approximately 800 bp (typically 500–600 bp with non-enriched DNA). The longer read lengths in Sanger methods display significant advantages over other sequencing methods especially in terms of sequencing repetitive regions of the genome. A challenge of short-read sequence data is particularly an issue in sequencing new genomes (de novo) and in sequencing highly rearranged genome segments, typically those seen of cancer genomes or in regions of chromosomes that exhibit structural variation.[15]
Applications of microfluidic sequencing technologies
Other useful applications of DNA sequencing include
Device design
The sequencing chip has a four-layer construction, consisting of three 100-mm-diameter glass wafers (on which device elements are microfabricated) and a polydimethylsiloxane (PDMS) membrane. Reaction chambers and capillary electrophoresis channels are etched between the top two glass wafers, which are thermally bonded. Three-dimensional channel interconnections and microvalves are formed by the PDMS and bottom manifold glass wafer.
The device consists of three functional units, each corresponding to the Sanger sequencing steps. The thermal cycling (TC) unit is a 250-nanoliter reaction chamber with integrated resistive temperature detector, microvalves, and a surface heater. Movement of reagent between the top all-glass layer and the lower glass-PDMS layer occurs through 500-μm-diameter via-holes. After thermal-cycling, the reaction mixture undergoes purification in the capture/purification chamber, and then is injected into the capillary electrophoresis (CE) chamber. The CE unit consists of a 30-cm capillary which is folded into a compact switchback pattern via 65-μm-wide turns.
Sequencing chemistry
- Thermal cycling
- In the TC reaction chamber, dye-terminator sequencing reagent, template DNA, and primers are loaded into the TC chamber and thermal-cycled for 35 cycles ( at 95 °C for 12 seconds and at 60 °C for 55 seconds).
- Purification
- The charged reaction mixture (containing extension fragments, template DNA, and excess sequencing reagent) is conducted through a capture/purification chamber at 30 °C via a 33-Volts/cm electric field applied between capture outlet and inlet ports. The capture gel through which the sample is driven, consists of 40 μM of oligonucleotide (complementary to the primers) covalently bound to a polyacrylamide matrix. Extension fragments are immobilized by the gel matrix, and excess primer, template, free nucleotides, and salts are eluted through the capture waste port. The capture gel is heated to 67-75 °C to release extension fragments.
- Capillary electrophoresis
- Extension fragments are injected into the CE chamber where they are electrophoresed through a 125-167-V/cm field.
Platforms
The Apollo 100 platform (Microchip Biotechnologies Inc., Dublin, CA)[16] integrates the first two Sanger sequencing steps (thermal cycling and purification) in a fully automated system. The manufacturer claims that samples are ready for capillary electrophoresis within three hours of the sample and reagents being loaded into the system. The Apollo 100 platform requires sub-microliter volumes of reagents.
Comparisons to other sequencing techniques
Technology | Number of lanes | Injection volume (nL) | Analysis time | Average read length | Throughput (including analysis; Mb/h) | Gel pouring | Lane tracking |
---|---|---|---|---|---|---|---|
Slab gel | 96 | 500–1000 | 6–8 hours | 700 bp | 0.0672 | Yes | Yes |
Capillary array electrophoresis | 96 | 1–5 | 1–3 hours | 700 bp | 0.166 | No | No |
Microchip | 96 | 0.1–0.5 | 6–30 minutes | 430 bp | 0.660 | No | No |
454/Roche FLX (2008) | < 0.001 | 4 hours | 200–300 bp | 20–30 | |||
Illumina/Solexa (2008) | 2–3 days | 30–100 bp | 20 | ||||
ABI/SOLiD (2008) | 8 days | 35 bp | 5–15 | ||||
Illumina MiSeq (2019) | 1–3 days | 2x75–2x300 bp | 170–250 | ||||
Illumina NovaSeq (2019) | 1–2 days | 2x50–2x150 bp | 22,000–67,000 | ||||
Ion Torrent Ion 530 (2019) | 2.5–4 hours | 200–600 bp | 110–920 | ||||
BGI MGISEQ-T7 (2019) | 1 day | 2x150 bp | 250,000 | ||||
Pacific Biosciences SMRT (2019) | 10–20 hours | 10–30 kb | 1,300 | ||||
Oxford Nanopore MinIon (2019) | 3 days | 13–20 kb[19] | 700 |
The ultimate goal of high-throughput sequencing is to develop systems that are low-cost, and extremely efficient at obtaining extended (longer) read lengths. Longer read lengths of each single electrophoretic separation, substantially reduces the cost associated with de novo DNA sequencing and the number of templates needed to sequence DNA contigs at a given redundancy. Microfluidics may allow for faster, cheaper and easier sequence assembly.[14]
See also
- Second-generation sequencing
- Third-generation sequencing
References
- S2CID 6384349.
- PMID 34346163.
- ^ PMID 21801614.
- ^ PMID 271968.
- S2CID 27800972.
We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.
- PMID 4000959.
- PMID 32070230.
- ISSN 0362-4331. Retrieved 2021-12-05.
- PMID 34548928.
- ^ "Phred - Quality Base Calling". Retrieved 2011-02-24.
- PMID 21245079.
- PMID 15514094.
- PMID 20203000.
- ^ S2CID 4851728.
- ^ PMID 18703132.
- ^ Microchip Biologies Inc. Apollo 100
- PMID 17623451.
- PMID 31096307.
- PMID 29273626.
Further reading
- Dewey FE, Pan S, Wheeler MT, Quake SR, Ashley EA (February 2012). "DNA sequencing: clinical applications of new DNA sequencing technologies". Circulation. 125 (7): 931–944. PMID 22354974.
- Sanger F, Coulson AR, Barrell BG, Smith AJ, Roe BA (October 1980). "Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing". Journal of Molecular Biology. 143 (2): 161–178. PMID 6260957.