What is a permutation?
A number of ways to organize items in a specific order. In our case, base pairs of DNA. Eg. Codons (AGC and TAG are different permutations)
How many distinct permutations in sequence can a stretch of a sequence 2 bases long have?
4 possibilities of base pairs (A,G,C,T) and a stretch of 2 bases. Therefore, 4^n = # of permutations.
So 4^2 = 16 different permutations in a stretch of 2 bases.
How do we determine the number of sites of n bases in a genome of size m?
if n = 1000
and m = 5.0x10^6
number of sites = m - (n-1)
(5.0x10^6) - (1000-1)
= 4.999x10^6
There are 4.999x10^6 sites of n = 1000 in a 5.0x10^6 base genome
Note: we assume this is single stranded DNA, so if double stranded, double your answer
How long does a DNA sequence have to be for us to expect it to be unique within a given genome?
We can expect a sequence to be unique in a genome when the number of permutations is greater than the number of sites of n bases in the genome.
How do we calculate n when the genome is known?
Bacterial genome is approx. 8x10^6 in bacteria. when is the number of permutations greater than the length of the genome? set 8x10^6 = 4^n then solve for n using logs
What are Universal Bits?
Universal bits refer to measuring DNA sequence information in terms of bits of information, rather than just counting base pairs. In DNA, the information content at any position in DNA is 2 bits (1 bp, 2 bases)
Ex. GGCCC = 5, so 2(5) = 10.
Information Theory: Minimum amount of bits to find our target object
The minimum amount of bits we need to find our target object is determined by the log2N equation. Ex. If N = 8, our minimum bits is 3. So we need to measure in 3 bit increments. (If there is only 1 bit present when we need 3, we will find additional targets to our selected target. We will not be able to accurately identify our target object.)
What is a BLAST Search?
Blast Stands for: Basic Local Alignment Search Tool. BLAST is a method used to compare DNA or protein sequences against a database to find similar sequences
How does BLAST Work?
You give BLAST a DNA or Protein Sequence and it searches a database to find: similar sequences, homologous genes, evolutionary relatives, or possible functions. BLAST looks for local regions of similarity, not full sequence matches.
BLAST: Query Strand
You break your sequence of interest, the Query, into shorter segments. These segments are then searched across the database where potential matches will be found.
What are the Two Main Statistics BLAST Search Gives?
Score and E-Value.
- Alignment Score = higher the score the better
- E-value = How likely this match occurred by chance (lower e-value the better)
Whata re the 4 Methods of DNA Sequencing?
Sanger (functions through chain termination and di-deoxy nucleotides)
Illumina (DNA sequencing by synthesis, uses fluorescence and photo imaging)
Nanopore (Assesses current change of nucleotides through pore)
PacBio (Uses DNA Pol and circularizes targets to track what bases are added wiht fluorescence)
Illumina Sequencing
Illumina uses fluorescently labelled nucleotides which stops elongation from occurring - images are taken after fluorescent nucleotides are added, then they are washed off, then repeat the cycle.
Step 1 and 2 of Illumina Sequencing
- Library Preparation
- Genomic DNA is fragmented into small pieces (mechanically or via. tagmentation: cut and tag DNA by transposomes)
- Adapter sequences are then added to each ends of the fragment with primer binding sites and flow cell binding regions (sometimes barcode regions if multiple samples being sequenced) - Cluster Generation (Bridge Amplification)
- DNA fragments are denatured into single strands which bind complementary oligos on the flow cell
- DNA pol the synthesizes a new strand and the old strand is washed away
- New strand bends over and its adaptor hybridizes to the second type of oligo
- DNA pol synthesizes a complementary strand and double strand is denatured into two copies attached to oligos
- this repeats many times forming a cluster of identical copies (amplifies fluorescence)
- Reverse strand is then cleaves and washed away
Steps 3 and 4 of Illumina Sequencing
- Sequencing by Synthesis
- Primer binds to the adaptor region
- Modified dNTPs are added and have fluorescent labels and 3’ reversible blockers
- Only one nucleotide is aded per cycle
- After cycle, fluorescent dye and blocker are chemically removed and next cycle begins
- Number of cycles = read length - Index Reads (if Multiplexing)
- After read 1, read product is washed away and index 1 primer is added
- Index read is generated and the index 2 read follows
- this allows for post-sequencing separation of pooled samples
Steps 5 and 6 of Illumina Sequencing
- Paired-End Sequencing
- After read 1, teplates are regenerated and the forward strands are washed away.
- Reverse strands remain and read 2 primer is adde so sequencing repeats from the opposite end
- Forward and reverse reads can be paired during analysis - Data Output and Analysis
- Hundred of millions/billions of reads
- Each cluster is one original DNA fragment
- Analysis separates reads by index, pairs forward and reverse reads, aligns reads to reference genome, and identifies variants (SNPs, insertions, etc.)
Pacific Bioscience Sequencing
Method which uses many different wells which immobilize DNA polymerase and the target DNA. Target is circularized and as each base is added it fluoresces which can be tracked by the microwells.
Steps 1 and 2 of PacBio Sequencing
- Library preparation
- DNA (or cDNA from RNA) is isolated
- Special hairpin adaptors are ligated to both ends which creates a circular DNA molecule
- Allows the DNA pol to sequence the forward and reverse strand (multiple times) - Single-Molecule Immobilization
- Load into SMRT cell which have thousands of tiny wells called ZMWs(Zero-Mode Waveguides)
- ZMWs are illuminated and have a detection window at the bottom
Steps 3, 4 and 5 of PacBio Sequencing
- Real-Time Sequencing
- Each nucleotide has a fluorescent label attached to the phosphate and emits light when incorporated
- When the light is detected the fluorescent tag is cleaved off and the polymerase continues - Circular Sequencing Advantage
- DNA is circular so polymerase can go around the circle, sequence forward and reverse strands, and repeat multiple passes
- This improves accuracy - Sequencing Modes
- Circular Consensus Sequencing: Polymerase goes around multiple times and reads same molecule repeatedly so it is very hgih accuracy. Produces shorter read lengths
- Continuous Long Read: Polymerase reads long fragment once and produces extremely long reads. Less accurate than CCS but useful for structural variation detection
Nanopore Sequencing
Method of DNA sequencing which assesses a change in current when nucleotides pass through a nuclear pore.
Steps of Nanopore Sequencing
- Single stranded RNA or DNA is threaded through the nuclear pore
- Disruption of the ionic current is measured in signal trace (each nucleotide disrupts the current in a unique and distinguishable way)
Comparison of 4 Methodologies
- Sanger is less efficient for many reads (1 at a time) but it is high quality/accurate. Very cheap
- Illumina is very efficient and high accurate. Uses two reads for each different fragment (forward and reverse) and you can get billions of reads per cycle. Very expensive.
- PacBio is efficient for long strands. More accurate than Illumina but less costly
- Nanopore is good for long strands of DNA/RNA molecules and can have millions of reads but reduced accuracy. Similar pricing to PacBio.
What is DNA Sequencing and DNA Sequence Alignment?
DNA Sequencing: The process of determining the exact order of nucleotides in a DNA molecule
DNA Sequence Alignment: The process of aligning two or more DNA sequences to identify regions that are similar or different
What is a Contig?
A continuous DNA sequence from a collection of overlapping sequences which will hopefully represent/span the whole genome.
