Genomics Flashcards
(16 cards)
- denature dsDNA
- Add primer, DNA polymerase, dNTPs and fluorescent-labeled ddNTPs
- DNA pol extends the primer by sequentially adding nucleotides that base pair with the template strand
- The occasional incorporation of a ddNTP terminates polymerization
- Electrophoresis of chain-terminated DNA molecules is done in a capillary gel
- Dye color tells you the base, size separation tells you the position of the base
Sample read length: 700-900bp
DNA Sequencing: Sanger Sequencing
sequencing reads that are identical from one clone to the next are overlapped to create a single contiguous sequence
contig
helps piece shotgun sequences together using restriction site mapping
1. Digest genomic DNA into many small pieces with restriction enzymes
2. Fragments cloned into a vector (plasmid)
Hierarchical genome sequencing and assembly
(Map-based sequencing)
- Cut many genome copies into random fragments
- Sequence each fragment
- Overlap sequence reads
- Overlap contigs for complete sequence
much faster than map-based assembly, but is more error-prone
Shotgun genome sequence assembly
- uses cyclic reversible termination with modified dNTPs (fluorescent tags)
- Adaptor sequences are ligated to genomic DNA fragments
- Adaptor sequences anneal to oligos on a flow cell
- Bridge amplification forms clusters of each fragment
- Sequencing primer anneals to adaptor to begin sequencing reaction
- Sequencing performed in cycles (1. incorporate nucleotide 2. take fluorescent image, 3. cleave fluorescent tag and reverse terminator)
- Number of cycles = length of sequencing read (35-250 bp)
- Paired-end reads repeat sequencing for other strand of DNA
Massively Parallel- Next generation sequencing (Illumina)
Used to build scaffolds
Reads overlap with different contigs, place adjacent contigs in order
Paired-end reads
Place assembled contigs in order
Scaffolds
Quality of assembly:
representation of each nucleotide in sequencing reads
coverage
Quality of assembly:
weighted median contig/ scaffold length
N50
uses changes in ion flow as DNA moves through a membrane pore to determine sequence
No DNA synthesis required
Long Read Sequencing - Nanopore
Order of genes on chromosomes of related species
synteny
Tools that allow us to define, identify, and catalog genome features computationally
Bioinformatics
Genome annotation: start with an initiation codon and end with a stop codon
can be identified by scanning all possible reading frames of a sequence for these features with a set length minimum
Open reading frames (ORFs)
Sequences that are descended from a common ancestor
homologous
arise by divergence through speciation events (homologs)
orthologs
arise through gene duplication events (homologs)
paralogs
