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.