Module 5.1 Sequencing By Ligation Flashcards
Sequencing by Ligation
features
- sequencing technique that involves use of ligases to identify nucleotide at given position in DNA strand
- relies on sensitivity of DNA ligase for base pairing mismatches to determine underlying sequence of target DNA fragment
- DNA ligase sensitive to structure of DNA and has lower efficiency when there are mismatches between bases of the two strands
- can ligate some mismatches to certain degree under certain conditions
DNA nick
single-strand break in a double-strand DNA molecule
T4 DNA ligase
- very efficient at sealing nicks and
joining or circularizing DNA fragments with blunt or cohesive ends - can ligate if containing one or more mismatches near ligation junction
- prone to ligate ends with mismatches
Taq DNA ligase
- only ligates nicks
- naturally able to discriminate against ligating substrates containing base pair mismatches and is considered to be higher fidelity than T4 DNA ligase.
ligation efficiency
factors
ligase type
temperature and salt concentration
- Higher temperature selectively reduces ligation efficiency of DNA ends with mismatches
position of mismatch
- further away mismatch is from ends of ligation, less discrimination it would be for ligation.
- When mismatch right at ligation position, the ligation efficiency is lowest
SoLiD sequencing
Library Preparation
- DNA fragmented to proper size.
- 5’ end = P1 Adapter, 3’ end = P2 adapter
- DNA fragment library amplified by emulsion PCR
- Beads enriched with clonally amplified DNA
- 3’ end (P2) modified so they can covalent bond in next step
- 3’ modified DNA-coupled beads deposited onto a glass slide (sequencing chip) and attach randomly
SoLiD sequencing
ligation probe
- 8 bases long (8-mer) with 3’ hydroxy group and 5’ fluorescent dye
- cleavage site between 5th and 6th nucleotide
- Starting from 3’ end, first 2 bases are specifically defined for each probe used for decoding nucleotides
- Bases 3-5 are degenerate sequences
- Bases 6, 7, and 8 are universal bases with dye attached to base 8
- Cleavage of fluorescent dye and bases 6-8 leaves a free 5’ phosphate group ready for further ligation.
- sequence denoted as 3’ XXNNNZZZ 5’
- 4x4 = 16 types of probes
- four fluorescent signals used, each representing a subset of the four dual based combos
Sequencing by Oligonucleotide Ligation and Detection
(SOLiD sequencing)
features
- uses sequencing by oligonucleotide ligation and 2 base encoding to generate sequencing data
- inherent proofreading function for reducing errors in the sequencing data
- 99.94% accuracy (relies on availability of a reference sequence)
- not hindered by homopolymers
- 35-50 bp read length
- 1-2 weeks to finish a run
- has issues with sequencing palindromic sequences
degenerate sequences
- nucleotides at these positions are randomly picked from any base ATGC
- sequences at these positions are not defined
- can be different in every oligo and are represented by N bases
- able to pair with matching nucleotides on template sequence
universal bases
- special type of base that can pair with any nucleotide
SoLiD sequencing
First Sequencing Round
Steps
- anneal universal primer to library molecule where primer 5’ end complentary to 3’ end of P1 adapter
- add the mixture of fluorescently labeled probes to template
- only one of the 16 oligos will be a perfect match and ligate. Other probes will bind to template but not be able to ligate with primer.
- probes not bound to primer washed away
- bound probe imaged
- empty primers without ligation extension deactivated by dephosphorylation
- bound probe cleaved between bases 5 & 6 to remove fluorescent group, leaves behind five-base ligated probe with 5’ phosphate ready for next round of ligation.
- Repeat cycle. 6th and 7th position are bases 1 & 2 for next probe. First 2 bases every 5 bases are sequenced.
- After 5-7 cycles = one round of sequencing
SoLiD sequencing
Sequencing Rounds 2-5
- need to perform a reset where initial primer and all ligated products are melted and removed
- new universal primer n-1 5’ end matches exactly one base before 3’ end of P1 adapter
- cycle 2 = queries positions 0 & 1 (instead of 1 & 2), then positions 5 & 6 (instead of 6 & 7) and so on
- after ligation cycles, extension products are removed, the templates are reset again.
- complete reaction of five rounds (n-2, n-3, & n-4 primers) with five cycles per round=25 bases of template from P1 end
2 base encoding
Error color pattern
single color change where all bases downstream of color change are different from reference sequence
2 base encoding
Single mutation color pattern
affect color code of two adjacent bases, resulting in two consecutive color changes
2 base encoding
Adjacent substitutions color pattern
two or three color changes
