Library preparation for Illumina sequencing
For the original SSV-Seq protocol including a PCR amplification step, DNA sequencing libraries were prepared from 2x1011vector genomes (2 replicates of 1x1011 vg) as determined by free ITR qPCR (D’Costa et al., 2016) and 200 ng of double-stranded DNA (dsDNA) as determined by spectrophotometry using the Nanodrop OneC (ThermoFisher Scientific, Waltham, MA). The complete SSV-Seq method is described in Lecomte et al. (Lecomte et al., 2019).
Three kits were tested for the PCR-free library preparation: KAPA HyperPrep kit (Roche, Basel, Switzerland), NxSeq AmpFREE Low DNA kit (Lucigen, Middleton, WI) and NEBNext Ultra II (New England Biolabs, Ipswich, MA). The PCR-free sequencing libraries were prepared following the instructions of the suppliers, at the exception of the purification steps and the adapter ligation. The adapters of the three kits were replaced by home-made Illumina-compatible P5/P7 adapters (Lecomte et al., 2019) and DNA purifications were carried out using the SPRIselect reagent (Beckman Coulter, Brea, CA).
The SSV-Seq 2.0 protocol was realized from 8x1011vector genomes (4 replicates of 2x1011 vg) of a rAAV vector batch. After DNA extraction and second-strand synthesis (Lecomte et al., 2019), the concentration of dsDNA was determined by fluorimetry using the Qubit 1X dsDNA High Sensitivity Assay kit (ThermoFisher Scientific, Waltham, MA). Two tubes per sample were prepared with 150 ng of DNA in a final volume of 100 µL TE10:1 pH8. DNA was sonicated using the Bioruptor UCD-200 (Diagenode, Liege, Belgium) and in accordance with the conditions described in Lecomte et al. (Lecomte et al., 2019) to reach an average target size centered on approximatively 300 bp. The fragmented DNA of the two tubes was pooled (300 ng) and purified using 1.6X of SPRIselect reagent (Beckman Coulter, Brea, CA). The magnetic beads with bound DNA were then washed two times with 360 µL of freshly prepared ethanol 80% and DNA was eluted in 20 µL of ultrapure DNase/RNase free distilled water (dH2O). Libraries were then prepared using the NxSeq AmpFREE Low DNA kit (Lucigen, Middleton, WI) that combined the end-repair and the A-tailing steps. The following mix was prepared in 0.2 mL PCR tube: 17 µL of previously sheared and purified DNA, 25 µL of 2X buffer and 8 µL of enzyme mix. The one-step reaction was realized using the Applied Biosystems Veriti Thermal Cycler (ThermoFisher Scientific, Waltham, MA, USA) for 20 min at 25°C with a 72°C heated lid, followed by a 20-min cycle at 72°C and hold at +4°C. Illumina-compatible P5/P7 adapters were prepared as described in Lecomte et al. (Lecomte et al., 2019) and diluted at 15 µM in dH2O. After DNA repair and A-tailing, 3 µL of diluted adapters and 4 µL of ligase were added in the 50-µL reaction volume. Adapter ligation took place in a thermocycler for 30 min at 25°C and was immediately followed by a double-1X SPRI purification. Each SPRI purification step includes two washes with 180 µL ethanol 80%. The first elution was performed in 50 µL of dH2O and the final elution was done in 16 µL of ultrapure distilled water.