Module 5.1 Sequencing By Ligation

Question 1

Sequencing by Ligation

features

Answer 1

• 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

Question 2

DNA nick

Answer 2

single-strand break in a double-strand DNA molecule

Question 3

T4 DNA ligase

Answer 3

• 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

Question 4

Taq DNA ligase

Answer 4

• 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.

Question 5

ligation efficiency

factors

Answer 5

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

Question 6

SoLiD sequencing

Library Preparation

Answer 6

• 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

Question 7

SoLiD sequencing

ligation probe

Answer 7

• 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

Question 8

Sequencing by Oligonucleotide Ligation and Detection
(SOLiD sequencing)

features

Answer 8

• 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

Question 9

degenerate sequences

Answer 9

• 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

Question 10

universal bases

Answer 10

• special type of base that can pair with any nucleotide

Question 11

SoLiD sequencing

First Sequencing Round

Steps

Answer 11

• 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

Question 12

SoLiD sequencing

Sequencing Rounds 2-5

Answer 12

• 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

Question 13

2 base encoding

Error color pattern

Answer 13

single color change where all bases downstream of color change are different from reference sequence

Question 14

2 base encoding

Single mutation color pattern

Answer 14

affect color code of two adjacent bases, resulting in two consecutive color changes

Question 15

2 base encoding

Adjacent substitutions color pattern

Answer 15

two or three color changes

Question 16

Combinatorial Probe Anchor ligation
(cPAL)

features

Answer 16

• detection of ligation products formed by anchor oligo hybridized to part of adapter sequence
• fluorescent degenerate sequencing probe containing specific nucleotide at interrogation position
• If nucleotide at interrogation position is complementary to nucleotide at detection position within target, ligation is favored and results in stable probe anchor ligation product that can be detected by fluorescent imaging
• ligation reaction condition optimized so ligase will ligate probes with higher efficiency when perfectly complementary to nucleic acid target region
• ligase fidelity decreases with more distance of acquired base from ligation end
• DNA template is unaltered after ligation product removed
• limited read lengths and slow sequencing

Question 17

Combinatorial Probe Anchor ligation
(cPAL)

process

Answer 17

• library molecules contains unique adapters flanking target DNA molecules
• anchor molecule hybridizes to one of unique adapters
• pools of 4 degenerate 9-mer oligos labeled with fluorophores for either ACGT in 1st position of probe
• correct matching probe is hybridized to template and ligated to anchor using T4 DNA ligase
• ligated product imaged
• ligate anchor probe molecules denatured
• process of hybridization, ligation, and imaging and denature repeated 5 times using new sets of probes containing known bases at positions 1-5
• can only read fluorescent signal when probe both hybridized to template and ligated to anchor
• need to reset reaction after 5 cycles (increased chance of incorrect pairing)

Question 18

cPAL reset reaction
(bases 6-10)

process

Answer 18

• degenerate anchor with five degenerate bases (NNNNN) anneals next to standard anchor and extends into target sequence
• degenerate anchor ligated to standard anchor = extended anchor
• 5-cycle sequencing repeats for next set of bases