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Summary Genome, proteome, metabolome analysis: definitions
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De nitionsGenome sequencing
Sanger Sequencing First generation sequencing method based on the selective
incorporation of chain-terminating ddNTPs during DNA replication.
Proces:
• DNA synthesis: DNA template + DNA pol + primer + ddNTPs
(labeled) + dNTPs are added in 4 di erent tubes (one for each
nucelotide). During DNA synthesis, ddNTPs can be incorporated
→ terminate = fragments of di erent lengths, all ending in the
same nucleotide
• Gel electrophoresis: separate the fragments based on size. Every
column contains a di erent nucleotide → read from bottom to top
Cycle sequencing Extension of the Sanger sequencing method. Uses thermostable
DNA pol (Taq) and labeled ddNTPs to sequence DNA.
Process
• Each reaction contains DNA template, Taq pol, primer, dNTPs and
1/4 ddNTPs (labeled) (again 4 reactions in di erent tubes)
• During temperature cycles: denaturation ds DNA → primer
binding → extension primer till ddNTP
• Separation by a capillary electrophoresis → detect the di erent
labels of the ddNTPs
Walking method sequencing Sequencing method used for longer DNA molecules. Stepwise
synthesis in 2 directions with new primers every 500 nt, based on
the sequenced part. Goes on until you have overlap and the gap is
closed
Shotgun sequencing Sequencing method used for longer DNA molecules. Fragment the
molecule randomly and sequence every fragment and make a
library (vector) → puzzle them all together based on the biggest
overlap (done in one direction walking method)
Hierarchial shotgun sequencing Sequencing method used for the whole genome. Genome is rst
broken down into large, overlapping fragments, often through the
creation of large-insert libraries, such as bacterial arti cial
chromosomes (BACs). These large fragments are then individually
sequenced using the shotgun sequencing method. Data from the
large fragments are assembled to create a preliminary draft of the
genome (often based on the large chromosomal tags from maps).
Next, gaps and ambiguities in the initial assembly are resolved.
Gaps are lled using the walking method with starting primers
anking the gap.
Whole genome shotgun Sequencing method used for the whole genome. DNA is randomly
fragmented into smaller, overlapping fragments. These fragments
are then individually sequenced. The resulting short sequence
reads are computationally assembled into longer contigs and
sca olds to reconstruct the complete genome sequence.
Genetic (linkage) genome map Identi cation and ordering of genes or genetic markers along a
chromosome based on their relative distances (measured in cM), as
determined by the frequency of genetic recombination events. In
linkage mapping, the closer two genes are on a chromosome, the
less likely they are to undergo recombination and segregate
independently.
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, Physical genome map Determining the physical positions and order of DNA sequences or
genes along a chromosome or a genome. Unlike genetic mapping,
which is based on recombination frequencies, physical mapping
provides information about the actual physical distances between
markers, often measured in base pairs.
• Can be done with restriction mapping: use 3 samples (RE1, RE2
and both) → gel electrophoresis
• Can be done with contig mapping: large fragments of genome →
gel electrophoresis → look for common bands → assemble tiling
path
Tiling path Set of overlapping DNA clones or fragments that span a speci c
genomic region, forming a continuous and non-redundant set of
sequences.
Combinational PCR Used to generate a diverse library of DNA fragments through the
combination of multiple primer sets in a single PCR reaction. In this
method, a pool of primers with various sequences is used to
amplify multiple target regions simultaneously. The primers are
designed to have unique combinations, allowing for the generation
of a complex mixture of DNA fragments that represent various
sequences from the template DNA.
Sca old After the initial assembly of contigs in whole genome shotgun
sequencing, gaps between them may still exist. Sca olding
involves placing and orienting contigs relative to each other to
create longer, more continuous sequences. The resulting sca olds
are valuable for understanding the overall structure of a genome,
including the arrangement of genes and other functional elements
Gene annotation Adding as much information as possible about the genome content
using the right prediction tools. Eg nding ORFs, promoters, and
preforming a homology search — used for gene identi cation in
eukaryotes
Codon usage Used in identi cation of genes. Check if it is a ORF by checking if
the frequency of the codon corresponds to the ones you know are
present → know if its a true protein coding sequence, not chance
CpG islands Used in identi cation of genes. Are high in GC content, often found
in promoter regions; their methylation state can cause changes in
gene expresion
EST (expressed sequence tag) Short DNA sequences derived from cDNA that represent partial
fragments of transcribed genes. ESTs are obtained through the
sequencing of cDNA libraries. Once sequenced, ESTs are aligned
to the genome or existing gene databases to identify and annotate
genes
Pyrosequencing NGS. Relies on the detection of pyrophosphate (PPi) during DNA
synthesis (PPi → ATP (by ATP sulfurylase) → light (by luciferase)).
(Add one type of dNTP, when complementary → light). Used in 454
sequencing — real time
454 sequencing DNA sample is fragmented into short sequences, and each
fragment is ligated to tiny DNA beads (involves addition of
adapters). These beads are then subjected to emulsion PCR to
amplify the DNA fragments on the surface of beads in water-in-oil
droplets → beads are loaded onto a picotiter plate →
pyrosequencing
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