Table 1 Optimized conditions for PacBio-LITS library preparation
Experimental conditions | Library insert sizes | |||
|---|---|---|---|---|
1 Kb | 4 Kb | 6 Kb | 7-8 Kb | |
Input DNA | 500 ng | 1 μg | 1.5 μg | 2 μg |
Shearing method | Covaris | Covaris or g-TUBE | g-TUBE | g-TUBE |
Shearing condition | 1 Kb shearing with microTube | Covaris: 5 Kb shearing with miniTube or g-TUBE: 12000 rpm, 45 sec. | 7000 rpm, 3 min. | 4800 rpm, 8 min. |
Size selection method | Pippin | Blue Pippin | Blue Pippin | Blue Pippin |
Size selection condition | Range mode: 900 bp-1.3 Kb | Range mode: 4 Kb-8 Kb | Range mode: 5 Kb-9 Kb | Range mode: 8 Kb-12 Kb |
DNA Polymerase | Phusion HF Polymerase | TaKaRa LA Taq Polymerase (Hot-start) | TaKaRa LA Taq Polymerase (Hot-start) | TaKaRa LA Taq Polymerase (Hot-start) |
Pre-cap PCR cycle # | 6 | 8 | 8 | 10 |
DNA amount into Capture | 1 μg | 1 μg | 2 μg | 2 μg |
Post-cap PCR cycle # | 14 | 16 | 16 | 18 |
Mean mapped subread length | ~700 bp | ~1.8-2 Kb | ~2-2.5 Kb | ~2.5-3 Kb |