Meridian’s Glycerol-Free NGS Library Preparation Solution Overview of library preparation by fragmentation and relevant Meridian products to perform each step. Fragments with 5’ and 3’ overhangs are repaired to create blunt ends, these are then phosphorylated and A-tailed, leaving ends amenable to adaptor ligation. Sequencing adaptors are then ligated to create complete sequencing-ready library fragments.
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
STEP 1 Fragmentation Physical or enzymatic
5’
5’
3’
3’
5’ 3’
5’ 3’
3’
5’
5’
3’
5’
5’
5’
5’
3’
3’
5’ overhang 3’ 3’
3’ overhang
5’ 3’ 5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
3’
3’
5’
5’
5’
5’
3’
3’
5’ overhang
5’ overhang
3’ overhang
3’ overhang
5’
5’
3’
3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
3’
5’ 3’ 5’ 3’ 5’ 3’ 5’
5’
3’
5’ 5’ 5’ 3’
5’ 5’ 5’ 3’
3’ 3’
3’ 3’
5’ overhang
3’ overhang
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
STEP 2
Create blunt ends
(3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction)
3’ 3’
5’ 5’
5’ 5’
3’ 3’
5’ 5’ overhang 5’ overhang 5’ overhang P 3’
3’ overhang 3’ overhang 3’ overhang 5’ 3’ 5’ 3’
Glycerol-Free T4 DNA Polymerase (HC) (MDX207) SEE PAGE 3 Glycerol-Free DNA Pol I Klenow Fragment (HC) (MDX208) SEE PAGE 3
5’
3’ 5’ 3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction)
5’ 3’
5’ 3’
5’
5’
3’
3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
(3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction)
P
P
5’
5’
3’
5’
P
3’
3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) 5’ 3’ 3’ 5’ P 5’ P
P 5’ (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) 5’ 3’
5’ 3’
5’ 3’
3’
3’
P
5’
STEP 3
Phosphorylation
A
Glycerol-Free T4 Polynucleotide Kinase (HC) (MDX206) SEE PAGE 4
3’
P P
5’ 5’
3’
3’
5’
P 3’
P 5’ 5’
3’ 3’
A
P A
A
5’
3’
3’ 3’ P 5’ P 5’ P 5’ A- 5’ A 3’ 3’ A 3’ -T A -T
P 3’
-A 5’ 5’ 5’ 5’ 5’ A A
P P 3’ P
3’ 5’
P
A
-T
5’
3’ 3’ P
STEP 4
A-tailing
A-
T- A 5’
P
Glycerol-Free Taq HS 50 U/ µ L (MDX170) SEE PAGE 5
Adaptor -A T- -A T-
3’ 3’
Adaptor
A A P 5’ A- 3’ A -T A-
P P
3’
Adaptor
Adaptor
Adaptor
Adaptor
-A
T-
5’
P
Adaptor
-T -T A-
Adaptor -A
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor
STEP 5
Adaptor ligation
A- A-
T- T-
-A
Glycerol-Free T4 DNA Ligase (HC) (MDX200) SEE PAGE 5
-T
-A
T-
Glycerol-Free High-Fidelity Pfu (HC) (MDX203) SEE PAGE 6 STEP 6 Library amplification
2
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