Sequencing Methods Flashcards
original sequencing methods
Sanger sequencing
-sequencing by synthesis
Maxam-Gilbert sequencing
-chemical degradation
sanger method:
primer incorporates radioactive label
termination by dideoxynucleotides (have H instead of 3’ OH - can no longer add new nucleotides)
dNTP - Deoxynucleotide TriPhosphate
ddNTP - DiDeoxy …
-denature plasmid (that has sequence of interest cloned into it)
- reanneal radiolabelled primer that hybridises next to sequence to be determined
4 reactions set up:
-each of 4 reactions includes a ddNTP of each base (ATGC)
-potential competition for incorporation of ddNTP vs dNTP
-at some frequency a ddNTP is incorporated
-chain extensions terminated at different lengths
-terminations occur at each of the 4 nucleotides in each reaction
-can fraction with PAGE
dye terminator sanger sequencing:
no radiolabelled dNTPs used
can be terminated with ddNTP with a fluorescent label (different for each base)
-all 4 reactions run in same lane of gel
-laser activated fluorescent label at bottom and fluorescence is detected and recorded
- order of bases can be ascertained from order of different dyes
how is whole genome sequenced with Sanger method?:
genome molecularly cloned as large fragments in BAC (bacterial artificail genome ) library
overlapping BAC clones identifies
each BAC is then cut into many smaller random fragments and “shotgun cloned” into plasmid vectors
-cloned DNA plasmids then sanger sequenced using a universal primer and final sequence assembled from known overlaps
NGS technologies?
next generation sequencing technologies
rely on methods that sequence DNA directly - no starting molecular cloning or PCR production
genomic DNA is reduced to millions of small random fragments representing all the sequences of the starting DNA (DNA fragment library)
these fragments are then simultaneously sequenced in a single procedure
so common themes in NGS:
-rely on massive parallel sequencing of fragments
-no separation of products by electrophoresis (unlike sanger)
-each reaction occurs on discrete solid phase clonal amplification of the single DNA molecule (polony/cluster)
-number of DNA copies in polony is enough to give detectable base-specific signals in sequencing reaction
-parallel reactions simultaneously monitored by detector system (e.g. fluorescence detector)
Illumina NGS method process?:
sequences DNA using DNA polymerase and fluorescent terminators
-sample preparation
-cluster generation - amplifies individual DNA molecules in situ
-sequencing by synthesis with simultaneous imaging to record fluorescence emission
-data analysis
process:
-short ss oligonucleotides of two defined sequences P5 and P7 are chemically bonded to glass slides/nanowells in a flow cell - forming a dense lawn
chemically bonded by 5’ ends, 3’ hydroxyl free pointing up
-preparing DNA: double stranded Adapters with same sequence as P5 and P7 oligonucleotides ligated to each end of the sonicated dsDNA fragments from genome of interest (200-300 nt)
adapters ligated in a way that gives different adapter sequences on each end of the fragment (one of each P5/P7)
-after ligation: dna population is denatured and added to glass slide
DNA attaches via complementary base pairing to bonded oligonucleotides
these oligonucleotides will act as primers
-density of DNA tuned so that DNA molecules attach at well separated posisitons
-DNA pol and dNTPs flushed though flow cell
each single DNA molecule is replicated in situ by dna polymerase, extending from the free 3’ end of the attachrd P5 and P7
-original DNA molecule is washed away after denaturation to leave a chemically bonded single copy of itself
–under renaturation conditions - this copy anneals to an adjecant chemically bonded complementary oligonucleotide primer (opposite to the one on its other end)
initiate another round of dna synthesis from this primer by DNA pol (“bridge amplification”)
-cycles of bridge amplification repeated many times (35 cycles)
in situ cluster of ~1000 identical copies in close proximity
clonal clusters (polonies?)
-the clusters contain two strands (one bound to the slide by either P5 or P7)
-one of the oligonucleotides is cleaved off the DNA and ddNTP is added to prevent further DNA synthesis from there
that strans is lost via denaturation
each cluster now contains a homogeneous population of strands for sequencing by the addition of a new primer
one end is covalently attached to one oligomer - the other hybridised via complementary base pairing
sequencing steps:
sequencing by synthesis from the recently added primer
-different fluorescent tag for dATP, dCTP, dGTP, dTTP
-These dNTPs have a block on their 3’ OH which prevents further elongation
this can be removed to free up the OH
-DNA pol added
-dNTP terminators added at adjacent position to the primer
i-mage records the laser activated fluorescence to see which base has just been added at each cluster/polony - recorded
-after imaging the fluorescent dye is chemically cleaved and the 3’ end is chemically unblocked by washing reagents through flow cell
this is repeated for every position on the sequence - fluorescence recorded for each
incorporation -> fluorescence detection -> cleavage of dye and terminator -> repeat for 30-50 nucleotides
how is illumina data processed?
same region of genome is represented by different overlapping clusters (sonication randomly breaking DNA)
depth of sequencing - no. of times the same region of genome has been sequenced
can get order of genome by organising these overlapping fragments
applications of Illumina?
-whole genome sequencing
-gene expression (sequencing cDNA)
-targeted re-sequencing
-ChIP sequencing
-microRNA discovery
methods for studying chromatin structure?
ChIP
see chromatin structure cards
chromatin structures are covalently cross-linked to the DNA with formaldehyde
DNase I hypersensitive sites to DNase I footprinting (see chromatin structure stuff)
can sequence results to find location on genome where different proteins bind
DNase I hypersensitivity of chromatin:
expose nuclei from cells with different levels of DNase I
then remove DNA from chromatin
cut with restriction enzyme
fractionate on gel
southern blot
open regions of chromatin more sensitive to DNase I than closed ones (euchromatin vs heterochromatin) and so fragments from those areas will be smaller
DNase seq analysis method
DNase I bounces off nucleosome heavy regions (inactive regions)
accesible to open region
cuts randomly many times
produces short DNA fragments in open chromatin regions
can add adapters to the DNAs generated
PCR amplify these fragments via the adapters
preferantially amplifies small fragments (longer ones more likely to fail)
map where the sequences of these fragments read on the genome
count number of sequence reads located to specific genome location
peaks at open, active regions of the genome
can use it to examine what genes are active in different cell lines (e.g. globin genes in different cells in haematopoietic system)
can correlate peaks from this to positions where ChIP finds TFs, activation markers
