- Materials
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- 50,000-60,000 target strand copies of gDNA
- Flow Cell Priming Kit (EXP-FLP002)
- Ligation Sequencing Kit (SQK-LSK109)
- Custom GSP UMI primers
- UVP oligos
- Consumables
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- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- g-TUBE™ (Covaris, Cat. # 520079)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Magnesium chloride (M1028)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Platinum™ SuperFi™ II Green PCR Master Mix (ThermoFisher, cat # 12369010)
- Quick Calf Intestinal Phosphatase (NEB cat #M0525)
- Thermolabile Exonuclease I (NEB, cat # M0568)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
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- Magnetic rack suitable for 0.2 ml PCR tubes
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Agilent Bioanalyzer (or equivalent)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Hula mixer (gentle rotator mixer)
- Thermal cycler
- Microfuge
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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For this protocol, we recommend starting with an input of 50,000-60,000 target strand copies.
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Ligation Sequencing Kit contents (SQK-LSK109)
Name Acronym Cap colour No. of vials Fill volume per vial (µl) DNA CS DCS Yellow 1 50 Adapter Mix AMX Green 1 40 Ligation Buffer LNB Clear 1 200 L Fragment Buffer LFB White cap, orange stripe on label 2 1,800 S Fragment Buffer SFB Grey 2 1,800 Sequencing Buffer SQB Red 2 300 Elution Buffer EB Black 1 200 Loading Beads LB Pink 1 360 -
Flow Cell Priming Kit contents (EXP-FLP002)
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200 -
Calculating input requirements
The more target strand copies input into the PCR, the more UMI clusters can be expected. However, this will in turn result in decreased cluster density. Therefore, we recommend an input of 50,000 — 60,000 target strand copies as an ideal compromise of total clusters and cluster density.
Worked example for the human genome:
- The haploid human genome is approximately 3.235 Gbp long
- The average DNA basepair has an atomic mass of 660 g•mol-1, therefore the atomic mass of the haploid human genome is 3.235E+9 bp * 660 g•mol-1 = 2.135E+12 g•mol-1
- To find the mass of a single copy of the haploid human genome, divide the atomic mass by Avogadro's constant: 2.135E+12 g•mol-1 / 6.022E+23 mol-1 = 3.545E-12 g or 3.545 pg.
- With a mass of approximately 3.545 pg, the dsDNA of the haploid genome contains complimentary top and bottom strands; half this mass represents a single target strand copy, therefore: 3.545 pg / 2 = 1.773 pg per target strand copy.
- To find the input mass of gDNA required: 50,000 target strand copies * 1.773 pg = 88,650 pg or 88.650 ng.
- In this instance, an input mass of 100 ng human gDNA will represent approximately 57,803 target strand copies, satisfying the input requirements of 50,000-60,000 target strand copies whilst remaining easy to quantify.