Ch. 8 - DNA Sequencing Flashcards
Genomics
Study of genomes as a whole rather than one gene at a time
Chain termination DNA sequencing, Sanger sequencing
The first methods for sequencing was developed in the 70’s (Sanger and Maxam-Gilbert sequencing). Both methods are based upon the production of a series of DNA subfragments (of all possible lengths) that have a specific 3’ end base, chain termination DNA sequencing. The fragments are separated on a gel with a column for each of the four possible bases at the 3’ end. A mixture of new nucleotides (dNTPs) and dideoxynucleotides (ddNTPs, which terminate elongation) are used when synthesizing the DNA fragments. Sanger sequencing is still used on shorter fragments of 600-1000 nt.
Automatic DNA sequencing and Dideoxysequencing
To synthesize DNA, a nucleotide needs to be added to the 3’-OH of the previous nucleotide. DNA polymerase can synthesize DNA strands, but only by elongating on a primer. Without a 3’-OH group the chain cannot be elongated. Dideoxynucleosides are missing the 3’-OH.
When a dideoxinucleoside labeled (on C3) with fluorescence is added to the growing chain, further elongation is not possible, and DNA synthesis is terminated. If the four different bases are labeled with four different fluorescent dyes, the termination/3’ end base of the fragment can be determined. Different ratios of ddNTP/dNTP are used when running a didexysequencing reaction to produce fragments of different lengths.
DNA fragments produced by DNA polymerase are of different lengths and is double-stranded. The fragments are separated by size using denaturing capillary electrophoresis. As the fluorescent DNA fragments migrate through a laser beam, a detector determines which base is located at the 3’ end.
DNA Sanger sequencing setup and automatic sequencing (BigDye)
- High copy DNA fragment (plasmid vector or PCR product), which is the template
- Sequencing buffer with Mg2+, cofactor for DNA polymerase
- Sequencing primer (determines sequencing initiation point, and hybridizes to the template
- dNTP mixed with ddNTP terminators (labeled with fluorescence for each base)
- DNA polymerase (normally thermostable)
All these components come in a kit (e.g. BigDye terminator cycle sequencing kit), except for the template DNA and the sequence primer.
Primer Walking
If cloned DNA fragments of over 1000 bp are to be fully sequenced, it is often necessary to run several sequencing reactions. New sequencing primers are deigned based on the new sequence information obtained from the first or previous primer (taken from the 3’ end of the newly known sequence). This is repeated until the entire fragment is sequenced. To be sure that the sequence is correct, both strands are usually sequenced.
Next Generation Sequencing (NGS)
Massive parallel sequencing that increases the output. NGS allows sequencing of millions of DNA fragments at the same time.
General steps that are common for many NGS technologies (ex. seq. of gDNA):
1. Library construction
2. Cluster generation
3. Sequencing
4. Data analysis
Library construction
- Library construction: gDNA is fragmented into smaller dsDNA pieces, either mechanically or enzymatically. Ultrasonic disruption can be used to fragment the DNA. The fragments can then be separated by by size through nebulization.
The ends of the DNA fragments are modified by ligation of adapters. This is done by adding T7 and Klenow DNA polymerases, which make blunt ends on the fragments. T4polynucleotide kinase the adds phosphate groups to the 5’ end of the double-stranded fragments. Klenow polymerase has terminal transferase activity, and adds adenines (A) to the 3’ ends. Adapters with T at their 3’ ends bind to the phosphate groups, and are ligated to the fragments by T4 DNA ligase. Adapters contain important features, such as barcode sequences, binding sites for sequencing primers and sequences complimentary to the solid support, which are used during the sequencing process. This allows amplification of the fragments through PCR, and to multiplex the sequencing.
Illumina: Cluster generation
- The DNA fragments (the library) are distributed at separate locations on a bead, a silica surface/nanowell (illumina), in a well or another solid surface: The nanowells in the Illumina system contain a lawn of small primers (oligonucleotides) that are complementary to the ends of the adapters, which were added to the DNA fragments in the previous step.
Once distributed, one DNA fragment per well or bead, the fragments can be amplified. In the Illumina system, bridge amplification is used. Each nanowell has the forward and reverse primers attached to the surface. One genomic DNA attaches to the nanowell. During annealing, both ends of the fragment attach to the surface primers, creating a bridge. After elongation, both ends of two strands are attached to the surface, before one end is released. Continued cycles create a cluster of fragments.
This produces up to 4 billion clusters containing about 1000 copies of the same template DNA.
Sequencing by reversible dye terminators synthesis
- The DNA in each of the locations is sequenced: DNA polymerase is attached to the end, not bound to the well surface, and synthesizes a complementary strand. The DNA templates are sequenced using reversible dye terminators (ddNTPs) that are labeled with fluorescent dye, one for each of the bases. The fluorescent dye can be removed. All four nucleotides are added at the same time to a flow cell channel together with buffer and DNA polymerase. The DNA sequencing primer is elongated with one ddNTP, which stops the elongation. The unbound nucleotides are washed away, and the flow cell is scanned. A computer detects the color and therefore which nucleotide is added. The fluorophore is removed, and elongation continues. The next nucleotide added is scanned in the same way, and so on.
Analyzing NGS data
- The sequence data is analyzed:
If samples are multiplexed: identify samples with the same index (from the same sample)
Up to 96 samples can be sequenced in one lane in the flow cell.
If the gDNA is sequenced and the genome is already known, all DNA reads are mapped to reference genome. Mapped reads are stored as binary alignment mapping files (BAM). A vcf format is used to specify differences from the reference sequence.
The Illumina technology can produce immense sequence data output (up to 96 samples in one run), 16 billion reads, each of 150 bp. However, this is costly: 10-100 of thousands kr for each run.
De novo sequencing
If the NGS data comes from an organism where the genome is completely unknown:
De novo assembly: a computer algorithm find as many overlapping DNA sequences as possible and try to assemble them in larger DNA sequences, contigs. The accuracy of the contigs/consensus sequence depends on read coverage (read depth, should be at least 30).
Ion Torrent DNA sequencing
First non-optical DNA sequencing method developed. Reads the DNA sequence by detecting H+ ions produced by the incorporation of nucleotides in a DNA strand by DNA polymerase. The release of H+ ions changes the pH in the well. The wells contain beads covered with DNA fragments. DNA fragments on the beads are amplified by emulsion PCR.
Beads with one DNA fragment is encapsulated in water droplets immersed in oil. Reagents needed for PCR are added, and emulsion PCR ensures that thousands of PCR fragments attached to beads are produced inside the “micro reactors”. dNTPs are added to the well, and the changes in pH are detected after each addition. The detection is done by an ion chip.
Sequencing takes only 2-4 hours
About 60-80 million reads.
Problems sequencing reads with AT or GC rich regions, and homopolymeric regions.
Single Molecule Real Time (SMRT) DNA sequencing
Pacific Biosciences SMRT DNA sequencing identifies the nucleotide added by DNA polymerase onto a growing strand of DNA by using fluorescently labeled pyrophosphate. Observes the DNA synthesis directly from a single DNA polymerase molecule.
DNA sequencing is performed in SMRT-chips, each of these containing 1 million zero-mode waveguides (ZMWs). A ZMW is a nanocontainer, which is optically transparent. It acts as a window and allows observation of a single DNA polymerase, attached to the bottom, as it adds nucleotides to a growing chain. Each nucleotide is labeled with fluorescence, one for each base, and emit a flash of light as they are added. The light is detected through the bottom of the ZMW.
Advantages of SMRT:
Long reads, can be more than 10 000 bp, makes it easier to assemble contigs (long stretched with continuous DNA sequence).
Disadvantage:
The accuracy of the DNA sequence from single reads are lower, important with sequence overlap.
Long and short reads
Sequenced DNA fragments
Long: SMRT, makes assembly of contigs long continuous DNA fragments easier, over 5-10 kbp
Short: Illumina, more complicated assembly, 75-400 base pairs
MinION nanophore sequencing
A protein nanophore is set in an electrically resistant membrane that permits a ssDNA to pass through, one at a time, and sequences it as it passes. This technology can procude ultra long DNA reads (up to 1 million bp), and a human genome was sequenced in 2018 using MinION nanophore sequencing. It measures 10 cm, and can be powered by the USB 3.0 port on a laptop.
The nanophore membrane separates two compartments of different charge, allowing negatively charged molecules like DNA can pass through in an extended conformation. While the DNA is passing through, a detector measures how much the current, due to normal ion flow is reduced. Since each base alters the current by different amounts, the detector can determine the sequence of the DNA strand as it passes through the pore, real time analysis.
