Development of a PCR-free protocol for the sequencing library preparation
Two parameters are critical for adapting the SSV-Seq library preparation protocol to a PCR-free method: (i) the reduction of the number of steps to avoid DNA loss during beads-based cleanup and (ii) the use of appropriate adapters (Figure 1 ). SSV-Seq adapters are suitable for use in a PCR-free protocol (Kozarewa et al., 2009) because they contain all elements required for the bridge amplification on Illumina flow cells, i.e. the sequences complementary to the flow cell oligonucleotides, the sequence targets of the P5/P7 sequencing primers and 6 base-indexes. Among PCR-free kits that are commercially available, 6 kits were first selected because of their compatibility with Illumina technology (Supplementary Table S3) . Three out of 6 kits were tested, i.e. KAPA HyperPrep (Roche), NxSeq AmpFREE Low DNA (Lucigen) and NEBNext Ultra II (New England Biolabs) based on the following criteria: (i) a low amount of fragmented DNA (≤ 200 ng) is required as input, (ii) the end repair and A-tailing steps are combined into a unique step, (iii) home-made adapters can be used and (iv) kits are compatible with Illumina paired-end sequencing. Libraries were prepared from 200 ng of fragmented PhiX174 DNA following the instructions of each kit, at the exception of two steps: the SSV-Seq adapters were used for the ligation instead of the commercial ones, and the post-ligation cleanup and size selection steps were replaced by a double purification with 1X SPRI beads (Figure 1, SSV-Seq 2.0 ). Libraries were prepared in triplicate to determine the robustness of each protocol. The efficiency of the 3 kits for generating sequencing libraries was compared qualitatively and quantitatively. DNA quality was controlled by high-sensitivity capillary electrophoresis (Figure 3a ). The electrophoregrams obtained on Agilent chip showed negligible amounts of free adapters in the final DNA libraries after the two SPRI purification steps whatever the kit used. Then, the number of adapter-ligated molecules in the libraries was quantified by qPCR Kapa assay using primers targeting the P5 and P7 sequences of the adapters, that correspond to the binding sequences to the Illumina flow cell (Figure 3b ). The ligation step of the kit NxSeq was the most efficient, leading to a library DNA concentration of 8.2 nM. For this reason, the NxSeq AmpFREE Low DNA kit was preferred over the two other kits and was included in the novel SSV-Seq 2.0 method for the PCR-free sequencing library preparation.
The optimized PCR-free protocol was then tested for the analysis of a rAAV vector batch, in parallel to the original SSV-Seq technique (Lecomte et al., 2019). The new protocol was applied to a purified rAAV8-CAG-GFP vector batch that was produced in HEK293 cells by plasmid transfection (Figure 1, PCR-free protocol ). The initial amount of rAAV vectors needed for the analysis and determined by free ITR qPCR (D’Costa et al., 2016) had to be increased from 2x1011vector genomes for the original SSV-Seq method to 8x1011 vg for the SSV-Seq 2.0. For each protocol, an additional replicate was used to control the second strand synthesis step. After DNA extraction and second strand synthesis, 300 ng of fragmented DNA, as determined by fluorometric quantification, was used as input for the PCR-free library preparation. The PCR-free library DNA, devoid of free adapters, was quantified by qPCR Kapa. The concentration of adapter-ligated fragments was on average 4.5 ± 0.2 nM in a final volume of 16 µL compared to a mean of 49.8 ± 5.1 nM in a final volume of 30 µL for the PCR-mediated protocol, which is above the minimal concentration required for Illumina sequencing. In conclusion, our optimized SSV-Seq 2.0 protocol allows to reach the quality and quantity of library DNA necessary for the analysis of AAV vectors by HiSeq Illumina sequencing.